Enzymatic Carbon–Sulfur Bond Formation in Natural Product

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Enzymatic Carbon−Sulfur Bond Formation in Natural Product Biosynthesis Kyle L. Dunbar,† Daniel H. Scharf,‡ Agnieszka Litomska,† and Christian Hertweck*,†,§ †

Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany ‡ Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216, United States § Friedrich Schiller University, 07743 Jena, Germany ABSTRACT: Sulfur plays a critical role for the development and maintenance of life on earth, which is reflected by the wealth of primary metabolites, macromolecules, and cofactors bearing this element. Whereas a large body of knowledge has existed for sulfur trafficking in primary metabolism, the secondary metabolism involving sulfur has long been neglected. Yet, diverse sulfur functionalities have a major impact on the biological activities of natural products. Recent research at the genetic, biochemical, and chemical levels has unearthed a broad range of enzymes, sulfur shuttles, and chemical mechanisms for generating carbon−sulfur bonds. This Review will give the first systematic overview on enzymes catalyzing the formation of organosulfur natural products.

CONTENTS 1. Introduction 2. C−S Bond-Forming Hydrolases 2.1. Endoproteases 2.1.1. Autoinducer Peptides 2.2. Thioesterases 2.2.1. Thiocoraline 2.3. (Ketosynthase-like) Acyltransferases 2.3.1. Calicheamicin 3. S-Transferases 3.1. S-Methyltransferases (SAM-dependent) 3.1.1. Bismethylgliotoxin 3.1.2. Collismycin 3.1.3. Echinomycin 3.1.4. Lincomycin A 3.1.5. Thiocoraline 3.1.6. Brassinin 3.1.7. S-Methylated RiPPs 3.2. S-Glycosyltransferases 3.2.1. Glycocins 3.2.2. Glucosinolates 3.2.3. Lincomycin A and Celesticetin 3.3. Glutathione-S-transferases 3.3.1. Glutathionyl and Cysteinyl Leukotrienes 3.3.2. Epipolythiodiketopiperazines 3.3.3. Glucosinolates 3.3.4. Allicin/Alliin 3.3.5. Pseurotin (trans to cis Isomerization) 3.4. S-Transferases Involved in Conjugate Additions

© 2017 American Chemical Society

3.4.1. Tropodithietic Acid and Roseobacticide A 3.4.2. Thienamycin 3.4.3. Leinamycin 4. C−S Bond-Forming Cyclases 4.1. Lanthipeptide Cyclases 4.2. NRPS Heterocyclization Domains 5. ATP-Dependent C−S Bond-Forming Enzymes 5.1. YcaO-like enzymes 5.1.1. Thiazoles and Thiazolines in RiPPs 5.1.2. Thioviridamide 5.2. Adenine Nucleotide Alpha Hydrolase Enzymes 5.2.1. 6-Thioguanine 5.2.2. Thiolactomycin 5.3. Adenylating Protein/Sulfur Carrier Protein Systems 5.3.1. Thioquinolobactin 5.3.2. BE-7585A 6. Oxygenases 6.1. Cytochrome P450 Monooxygenases 6.1.1. Camalexin 6.1.2. Cyclobrassinin and Spirobrassinin 6.1.3. Thienodolin 6.1.4. Griseoviridin 6.1.5. Ustiloxin

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Special Issue: Unusual Enzymology in Natural Products Synthesis

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Chemical Reviews 6.2. Nonheme Iron-Dependent Enzymes 6.2.1. Penicillins and Cephalosporins 6.2.2. Ergothioneine and Ovothiol 6.3. Flavoenzymes 6.3.1. Coelimycin 6.3.2. Sulfadixiamycins 6.4. Tyrosinases 6.4.1. Pheomelanins 6.4.2. Grixazones 6.4.3. Grape Reaction Product 7. Radical S-Adenosylmethionine Enzymes 7.1. Thioether-Forming rSAMs 7.1.1. Albomycin 7.1.2. Sactipeptides 7.1.3. γ-Subunit of Quinohemoprotein 8. Nonenzymatic C−S Bond Formations 8.1. Nonenzymatic Conjugate Addition 8.1.1. Enediyne Warhead Activation 8.1.2. Urdamycin E and BE-7585A 8.1.3. Ralfuranone D 8.2. Nonenzymatic Addition 8.2.1. Cyslabdan 8.2.2. Paulomycin S-Conjugates 8.3. Photochemical and Radical-Mediated Thioconjugation 8.3.1. Panphenazines 9. Conclusions and Outlook Author Information Corresponding Author Notes Biographies Acknowledgments References

Review

sulfur moieties that are pivotal for biological function (Figure 1), and, as might be expected, varied enzymatic mechanisms have evolved for the formation of the carbon−sulfur bonds in these molecules. Over the past decade, our understanding of enzymatic C−S bond formations in natural products has dramatically improved. Studies at the genetic, biochemical, and chemical levels have elucidated the sources of sulfur, the reaction mechanisms, and the types of biocatalysts involved. As in primary metabolism, persulfidic sulfur (R−S−SH) and thiocarboxylate groups on sulfur-donor proteins represent major ionic sulfur sources. In addition, thiols of cysteine and glutathioneand even sulfur dioxidemay serve as S-donors in secondary metabolism. Besides substitution reactions with sulfur nucleophiles, (conjugate) additions, and radical reactions are frequently observed. The aim of this Review is to provide the first overview of the current knowledge on biosynthetic pathways leading to sulfurbearing natural products. To systematize the many avenues leading to covalent C−S bonds, the Review is organized into sections on the different classes of biocatalysts involved. The reader will note that in some cases (e.g., glycosinolates, lincomycin, thiocoralin, gliotoxin, and phytoalexins) the biosynthetic pathways were dissected as multiple C−S bondforming enzymes are involved during biosynthesis. In some sections, C−S linkages of unknown biosynthetic origin are discussed. Beyond these open questions, it has not been possible to assign an exact reaction mechanism for the formation of every C−S bond; thus, some examples were only tentatively assigned to a particular section. Finally, select nonenzymatic C−S bond formations in secondary metabolism are highlighted.

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2. C−S BOND-FORMING HYDROLASES In both primary and secondary metabolite pathways, thioester bonds play an important role. Activated biosynthetic building blocks and intermediates are often provided as thioesters bound to coenzyme A and phosphopantetheinylated carrier proteins.10,12 In these cases, their formation typically requires ATPdependent ligases, and frequently transesterifications are observed. It is common knowledge that such thioesters are energy-rich moieties that are easily hydrolyzed or transformed into the corresponding esters or amides. However, there are remarkable biosynthetic pathways where thermodynamically stable esters and amides are converted into high-energy thioesters. Even more surprising is the fact that these energetically disfavored reactions are mediated by enzymes that belong to the hydrolase family: biocatalysts known to cleave amide and ester bonds.

1. INTRODUCTION Sulfur is a ubiquitously distributed element that is essential to all known living species. According to the iron−sulfur world hypothesis, it even played a key role in the evolution of life.1 Numerous essential biochemical processes in prokaryotic and eukaryotic cells are tightly linked to this particular element. Enzymes not only harbor iron sulfur clusters but also depend on cofactors that contain sulfur, such as thiamine, molybdopterin, biotin, and lipoic acid.2 Furthermore, many biosynthetic building blocks are activated as coenzyme A thioesters, and important detoxification processes, such as the conversion of cyanide to isothiocyanate3 and the conjugation of electrophilic toxins to glutathione and related compounds,4 depend on sulfur. Thio modifications are also important for stabilizing the tRNA structure and for accurate and efficient translation.5 Sulfur’s prime position in primary metabolism is indisputable, which is reflected by a large body of knowledge that has been presented in various review articles.6−9 In contrast, the role of sulfur in natural product biosynthesis has been somewhat neglected. One may recall that intermediates of fatty acids, polyketides, and nonribosomal peptides are tethered to phosphopantetheinylated carrier proteins through thioester bonds,10 as well as that S-adenosylmethionine is an important cofactor of methylations.11 Even so, apart from cysteine and methionine residues in peptides, secondary metabolites are mainly composed of carbon, hydrogen, oxygen, and nitrogen whereas sulfur atoms are scarce. However, there are innumerable examples of natural products containing diverse

2.1. Endoproteases

Proteases are ubiquitous in nature and are central to many biological functions. While most proteases catalyze the hydrolytic cleavage of amide linkages, a subset of proteases has been characterized that catalyzes ligation reactions. Such proteolytic ligases resolve the acyl-enzyme intermediate with a nonsolvent nucleophile to afford the ligated product. Ligating proteases are critical for biological processes, including the anchoring of proteins to the bacterial cell wall by sortase and peptidoglycan biosynthesis by penicillin binding proteins.13,14 In addition to the roles that ligating proteases play in primary cellular pathways, select family members are also used in natural product biosynthesis.15−19 These members catalyze the macro5522

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Figure 1. Representative sulfur-bearing natural products.

There are four known AIPs produced by S. aureus strains. The peptides vary in length (7−9 amino acids) and in sequence but are linked by the presence of a conserved thiolactone macrocycle spanning five amino acids.21 Genetic analyses revealed that AIPs are members of the ribosomally synthesized and posttranslationally modified peptide (RiPP) natural product class.20,22 AIPs are biosynthesized from a larger precursor peptide (AgrD) by the iterative action of two proteases (Scheme 1). First, the C-terminal tail of the peptide is removed (herein referred to as a follower peptide) by AgrB, a transmembrane cysteine endoprotease.23,24 After thiolysis of the peptide bond, the enzyme-bound thioester intermediate is resolved by the internal cysteine of AgrD to generate the thiolactone macrocycle. Although this enzyme-bound thioester has never been directly observed, mutation of the AgrD cysteine residue to a serine was found to cause a buildup of covalently linked AgrD and AgrB.25 Because this cross-linked species was not observed with a catalytically inactive version of

cyclization of a peptide substrate, thus endowing the natural product with a more rigid structure and enhanced proteolytic stability. Because the first step of such proteolytic macrocyclizations is the cleavage of an amide bond, it is perhaps not surprising that most characterized members ultimately catalyze the formation of an enthalpically neutral macrolactam linkage. One notable exception is found in the biosynthesis of peptidic quorum-sensing molecules. 2.1.1. Autoinducer Peptides. Autoinducing peptides (AIPs) are important quorum-sensing molecules that are widely produced by Gram-positive bacteria. In Staphylococcus aureus, AIP is the signal peptide of the accessory gene regulator (agr) system where its recognition initiates a signal cascade that leads to the global expression of virulence factors.20,21 Because of the essential role that this quorum sensing system plays in pathogenesis, AIP has been the subject of extensive investigation. 5523

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biosynthetic gene cluster demonstrated that the thioester linkage is formed between a Cys residue on the precursor peptide and an indolic acid derivative (Scheme 2).31 Notably, a member of the alpha−beta hydrolase superfamily (NosK) is predicted to install the thiolactone ring; however, this proposed function requires experimental verification.

Scheme 1. Thiolactone Formation Catalyzed by Transmembrane Cysteine Endoprotease AgrB Is a Key Step in the Biosynthesis of Autoinducing Peptides (AIPs) (LP, Leader Peptide; FP, Follower Peptide)

2.2. Thioesterases

Many polyketides, peptides, and hybrids thereof are produced by multimodular type I polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) that resemble miniaturized assembly lines.32 A hallmark of these so-called thiotemplate systems is that the growing intermediates are covalently tethered as thioesters to thiolation (T) domains, i.e., acyl or peptidyl carrier proteins (ACPs or PCPs, respectively). Thioesterase (TE) domains are typically located at the end of the assembly lines and are responsible for off-loading the mature polyketide/peptide chains.32,33 The full-length product is first transferred from the T domain onto a serine residue of the TE domain. Second, depending on the nature of the TE domain, the ester bond may be hydrolyzed to yield the linear product or cyclized into a lactone or lactam ring by intramolecular attack of an amino or hydroxyl group, respectively. While many examples of lactone- and lactamforming TE domains have been discovered, it was only recently that a thiolactone-forming TE domain was identified. 2.2.1. Thiocoraline. This peculiar C−S bond-forming reaction is involved in the biosynthesis of thiocoraline, a sulfur-rich metabolite of marine actinomycetes. Thiocoraline belongs to the family of chromodepsipeptides, which comprise several nonribosomally produced pseudosymmetrical peptidolactones and peptidothiolactones.34 Chromodepsipeptides are equipped with heteroaromatic substituents that enable DNAbis-intercalation, endowing many members of this family with cytotoxic, antiviral, and antibiotic activity. Thiocoraline is a promising anticancer agent and is structurally intriguing, as it is composed of two tetrapeptide chains fused into a bicyclic system by one disulfide and two thiolactone bonds (Scheme 3). Analysis of the thiocoraline (tio) biosynthetic gene cluster indicated that the tetrapeptide chains are assembled by an iteratively acting tetramodular NRPS (TioR and TioS) and implicated a TE domain in the macrothiolactonization.35 The proposed macrothiolactonization reaction was successfully reconstituted using the heterologously produced thiolation− thioesterase didomain (T−TE) of module 4 and synthetic substrates.36 As predicted, the T−TE didomain catalyzed both the ligation of tetrapeptidyl−thioesters and the subsequent

AgrB (Cys84Ser) and was sensitive to pH and temperature, this species is believed to be the enzyme-bound thioester intermediate. Following thiolactone formation, the peptide is secreted from the cell and the N-terminal leader peptide is removed by the signal peptidase, SpsB, to afford the mature natural product (Scheme 1).26 While these studies provided a mechanism for the biosynthesis of AIP, they did not provide an explanation for how AgrB catalyzes this thermodynamically unfavorable reaction. Reconstitution of liposome-incorporated AgrB in vitro demonstrated that the reaction does not require an exogenous energy source, ruling out the coupling of thiolactone formation with ATP hydrolysis.27 Rather, the thermodynamic sink is overcome by the partitioning of the thiolactone intermediate into the membrane and rapid proteolytic degradation (t1/2 ≈ 10 s) of the follower peptide. Apart from AIP, thiolactone macrocycles are also present in nosiheptide and glycothiohexide, members of the thiopeptide subclass of RiPPs.28−30 The discovery of the nosiheptide Scheme 2. Structures of Thioester-Containing Thiopeptides

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macrocyclization of the octapeptidyl intermediate. In the presence of a substrate analogue with a hydroxyl nucleophile in lieu of the Cys thiol group, macrolactones instead of the native macrothiolactones were obtained.

Scheme 3. Macrothiolactonization Mediated by a Thioesterase Domain of the Thiocoraline Assembly Line

2.3. (Ketosynthase-like) Acyltransferases

Canonical ketosynthases catalyze the Claisen condensation of acylthioesters and malonylthioesters to produce a diverse array of natural products.32,37 Recent work has demonstrated that select ketosynthase homologues function as acyltransferases that form ester or amide linkages rather than C−C bonds.38−43 Such an enzyme has been proposed to form a thioester in calicheamicin biosynthesis. 2.3.1. Calicheamicin. Formation of the thioester in calicheamicin, the DNA-cleaving enediyne, likely involves an unusual type of acyltransferase. Analysis of the calicheamicin (cal) biosynthetic gene cluster of Micromonospora echinospora ssp. calichensis revealed a gene (calO4) that was initially believed to encode a ketoacyl-ACP synthase.44 Homologues of CalO4 are involved in the biosynthesis of glycosidic natural products such as chlorobiocin (CloN2),45 chlorothricin (ChlB3),46 and cervimycin (CerJ).47 In all cases, sugar residues are linked to rare building blocks via ester bonds. A phylogenetic analysis showed that CalO4 falls into a clade of ACP shuttle enzymes that are closely related to FabH-like enzymes, ketosynthase III homologues involved in the priming of fatty acid and polyketide synthases.47 Even so, functional analyses in vivo and in vitro unequivocally demonstrated that these enzymes function as acyltransferases that load activated acyl groups onto hydroxyl substituents of sugars. By analogy, it appears plausible that CalO4 transfers the acyl residue onto a thiol group of a thiosugar (Scheme 4). The thiol group of the sugar building block may be introduced by attack of a sulfur nucleophile onto a carbonyl group, followed by a lyase reaction.48 However, the proposed mechanism for the transacylation and the biosynthetic origin of the thiosugar require verification.

3. S-TRANSFERASES The biosynthesis of many sulfur-bearing natural products involves designated S-transferases, which catalyze the attack of a thiol to an activated carbon without the need of ATP. This reaction is similar to the thioester formations described in section 2 but does not involve the cleavage of activated ester or thioester moieties. There are two types of S-transferases, Smethylstransferases and S-glycosyltransferases, which are better known for their ability to form C−O, C−N, and C−C bonds using S-adenosylmethionine (SAM) and activated sugars as cosubstrates.11,49,50 The third S-transferase presented herein, glutathione-S-transferase, loads thiol groups onto different types of activated carbons. In addition to these three wellcharacterized enzyme families, there are several S-transferases that do not fall into these categories but share the ability to promote Michael additions of thio nucleophiles to diverse α,βunsaturated acceptor systems.

Scheme 4. Proposed Mechanism of Thioester Formation in Calicheamicin Biosynthesis

3.1. S-Methyltransferases (SAM-dependent)

From a chemical perspective, methylation of a thiol represents one of the simplest types of C−S bond formations. It is textbook knowledge that S-methylation is the final step in methionine biosynthesis from homocysteine.51 This reaction is often catalyzed by a cobalamine-dependent methionine synthase, which utilizes 5-methyltetrahydrofolate (N 5 MeTHF) as the methyl donor. SAM-dependent S-methylations 5525

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are also found in thiopurine detoxification pathways and as posttranslational modifications.50,52 In addition, the stepwise Smethylation of sulfide is a possible route to dimethylsulfide (Scheme 5), the most abundant organosulfur compound in the Scheme 5. SAM-Dependent Methylation of Hydrogen Sulfide and Organic Thiols

Figure 2. Crystal structure of TmtA in complex with Sadenosylhomocysteine (PDB entry code: 5EGP).

atmosphere, which is often referred to as the “smell of the sea”. Yet, this route has only been demonstrated in vitro with cellfree extracts of the ciliate Tetrahymena thermophila using SAM and sulfide or methanethiol as substrates.53 Although SAMdependent O-, N-, and C-methyltransferases are frequently associated with natural product biosynthetic pathways, until recently no genuine enzyme for a C−S bond formation was known for secondary metabolites. 3.1.1. Bismethylgliotoxin. An unusual S-methyltransferase involved in natural product modification was discovered in the context of a self-resistance mechanism against gliotoxin, a potential virulence factor of the human-pathogenic fungus Aspergillus f umigatus.54 Gliotoxin exerts its damaging effects through protein conjugation and redox-cycling, which requires the formation of free thiol groups. Two independent mechanisms confer resistance toward gliotoxin. First, the epidithiol may be oxidized into the dithio bridge by a flavin adenine dinucleotide (FAD)-dependent oxidase (GliT);55 deletion mutants of gliT showed significant higher sensitivity toward gliotoxin in comparison to the wild type.56 Second, long-term cultures of A. f umigatus produce bismethylgliotoxin (Scheme 6), which indicates that the thiol groups could be

are known, it seems that this back-up protection strategy is widespread among ETP-producing fungi. Notably, a similar scenario has been proposed for bacteria, specifically for holomycin biosynthesis and resistance in Streptomyces clavuligerus. Formation of the cyclic ene−disulfide involves a thioredoxin oxidoreductase-like enzyme (HlmI) that is homologous to GliT, and a hlmI deletion mutant produces Smethylated holomycin derivates.60 However, the corresponding enzymes catalyzing the S-alkylation have not yet been identified. 3.1.2. Collismycin. Whereas S-methylation of gliotoxin may be regarded as a detoxification pathway, several S-methyltransferases (S-MTs) are involved in the assembly of biologically active natural products. Collismycins are rare 2,2′bipyridyl natural products produced by a Streptomyces sp. that were initially identified as inhibitors of dexamethasone− glucocorticoid receptor binding.61 Since then, neuroprotective, antibacterial, and antifungal bioactivities have been attributed to the collismycins.62−64 2,2′-Bipyridyl natural products are biosynthesized by a hybrid PKS/NRPS from malonyl-CoA, lysine, cysteine, and leucine (Scheme 7).65−67 Following the formation of the bipyridyl ring system, the terminal leucine residue is cleaved by an amide hydrolase and the newly formed carboxylic acid is converted to an oxime by a multienzyme pathway. Although the collismycin biosynthetic pathway has not been fully elucidated, a SAMdependent S-methyltransferase is known to play a critical role. When a putative methyltransferase (clmM1) in the collismycin A producer Streptomyces sp. CS40 was inactivated, the resultant strain was unable to produce collismycin A but instead produced collismycin SN (Scheme 7).65 This intermediate was successfully converted into the corresponding S-methylated derivative, collismycin SC, by a mutant strain lacking the downstream-acting amide hydrolase, ClmAH. On the basis of these results, collismycin SN was proposed to be the product of bipyridyl ring formation, and ClmM1-catalyzed S-methylation was predicted to facilitate the cleavage of the S−N bond to yield collismycin SC.65 An alternative interpretation of the data is that S-methylation is required for recognition of collismycin SC by ClmAH and that collismycin SN is a byproduct formed from the spontaneous oxidation of the free thiol intermediate. When fed to the ClmAH-deficient strain, the reduction of collismycin SN would provide the free thiol for S-methylation by ClmM1. While it is clear that ClmM1 is responsible for Smethylation in collismycin A maturation, further work will be required to determine the exact biosynthetic route to collismycin A.

Scheme 6. Enzymatic Inactivation of the Reactive Epidithiol Form of Gliotoxin: (Top) GliT-Mediated Oxidation into the Epidithio Bridge (Impermanent) and TmtA-Catalyzed bis-SMethylation (Permanent); (Bottom) Analogous Route for Holomycin Processing

permanently inactivated by bisalkylation. In vitro and in vivo experiments showed that the S-methyltransferase TmtA, which is not encoded in the gli gene locus, is responsible for gliotoxin S-methylation.57,58 Analysis of the crystal structure of TmtA in complex with S-adenosylhomocysteine showed that one substrate and one cofactor binding pocket are present in each monomer (Figure 2), which suggested that the bisthiomethylation of gliotoxin occurs sequentially.59 Because many dimethylated epipolythiodioxopiperazine (ETP) derivatives 5526

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Scheme 7. Proposed Pathway for S-Methylation in Collismycin Biosynthesis; Two Routes for ClmM1-Catalyzed S-Methylation Are Shown

3.1.3. Echinomycin. An S-methyltransferase that rearranges a disulfide bond has been discovered in the context of echinomycin biosynthesis. Echinomycin islike thiocoraline (see section 2.2.1)an important member of the chromodepsipeptide family and can also intercalate into duplex DNA.34 Echinomycin differs from thiocoraline mainly in the pair of quinoxazoline residues and the substitution of the thiolactone moieties for lactones. In addition, echinomycin features a rare thioacetal bridge, which endows the bicyclic core with greater stability compared to the labile disulfide bond. Biosynthetic studies with the echinomycin producer Streptomyces lasaliensis revealed that this unique chemical motif is obtained by conversion of the disulfide bond of triostin Aand that this reaction is catalyzed by a single SAM-dependent methyltransferase (Ecm18) (Scheme 8).68 The crystal structure of Ecm18 in complex with echinomycin and S-adenosylhomocysteine (Figure 3) indicated that the disulfide−thioacetal conversion occurs in two stages. First, one sulfur atom is methylated; then the carbon adjacent to the sulfonium is deprotonated, yielding an ylide that rearranges into the thioacetal via a charge-delocalized transition state (Scheme 8).69 This model is supported by the finding that a singly Smethylated product is produced when the reduced form of triostin A is used as a substrate in the Ecm18 enzyme assay. 3.1.4. Lincomycin A. Besides formation through disulfide conversion, a thioacetal may be formed by alkylation of an anomeric thiol group as in the biosynthetic pathways leading to lincosamide antibiotics. This small family of ribosome-targeting compounds comprises lincomycin A, Bu-2545, and celesticetin (see section 3.2.3), each bearing an unusual 1-thiooctose sugar (Scheme 9).70 Early biosynthetic studies demonstrated that the methylthiolincosamide and propylhygric acid moieties of lincomycin A are produced independently and then linked; however, a biosynthetic route for the incorporation of the αlinked methylthiol moiety could not be provided.48,71−75 Only recently, the highly complex pathway has been investigated in detail (for sulfur incorporation route, see section 3.2.3). In the course of these studies, an S-methyltransferase (LmbG) was discovered that alkylates the thiol group of the octose (Scheme 9).76,77 3.1.5. Thiocoraline. A bifunctional enzyme with Smethyltransferase and amino acid adenylation activities has recently been identified that is involved in thiocoraline

Scheme 8. Conversion of a Disulfide Bond to the Thioacetal Group of Echinomycin by a SAM-Dependent Methyltransferase

biosynthesis (see section 2.2.1). In addition to the unusual thiolactone bonds, thiocoraline contains two extraordinary Smethylated L-cysteine units. Gene inactivation and biochemical analyses showed that TioN plays an essential role in the biosynthesis and activation of this rare amino acid.78 TioN is a stand-alone enzyme harboring an adenylation domain that is interrupted by the conserved SAM-binding region of SAM methyltransferases. TioN is remarkable in that it is the only interrupted, stand-alone A domain identified to date; all other 5527

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Scheme 10. Activation and S-Methylation of Cysteine to Produce and Incorporate the Rare S-Methyl Cysteine Residues of Thiocoraline

Figure 3. Crystal structure of Ecm18 in complex with echinomycin and S-adenosylhomocysteine (SAH; PDB entry code: 4NEC). The histidine residue thought to be responsible for the deprotonation of the β-carbon is displayed.

interrupted adenylation domains are components of NRPS modules.79 In vitro studies revealed that TioN adenylates Lcysteine, S-methylates the thiol group, and loads the amino acid onto its cognate thiolation (T) domain in module 4 (Scheme 10).78 Kinetic characterization of TioN demonstrated that the catalytic efficiency of the adenylation reaction is ∼500 times greater for L-Cys than for S-Me-L-Cys, indicating that adenylation precedes methylation. While TioN was able to catalyze methylation prior to or after T domain loading in vitro, the order of methylation and loading is not known in vivo. 3.1.6. Brassinin. Brassinin is a plant defense compound produced by cruciferous vegetables. This important member of the indole sulfur phytoalexin class of natural products has antifungal and anticancer activities and is a key intermediate in the biosynthesis of additional indole sulfur phytoalexins (section 6.1.2).80,81 Labeling experiments demonstrated that brassinin is biosynthesized from indole glucosinolate, likely through an isothiocyanate intermediate.82 Leveraging the observation that phytoalexins from Brassica rapa are only produced upon pathogen exposure, a recent study partially characterized the brassinin biosynthetic pathway.83 Plant leaves were challenged with Pseudomonas syringae pv maculicola and RNA sequencing was performed to identify genes that were upregulated following exposure. Among others, the expressions of a pyridoxal 5′-phosphate (PLP)-dependent C−S lyase (SUR1) and a SAM-dependent methyltransferase (DTCMT.a) were increased. Reconstitution of both enzymes in vitro demonstrated that SUR1 converts indole isothiocyanate cysteine to indole dithiocarbamate, which is subsequently methylated by DTC-MT.a to yield brassinin (Scheme 11). This biosynthetic proposal was further verified when brassinin was produced by tobacco leaves coexpressing the B. rapa glucosinolate and brassinin biosynthetic genes.

A SUR1 homologue (AtSUR1) is found in Arabidopsis thaliana, where it was previously implicated in glucosinolate biosynthesis (section 3.3.3).84 Notably, reconstitution of the activity of AtSUR1 in vitro demonstrated that it can use both cysteine−isothiocyanate conjugates and S-alkyl thiohydroximates as substrates.83 While further studies will be required to understand the remarkable substrate promiscuity of SUR1, the current data demonstrate that the enzyme plays a central role in both glucosinolate and phytoalexin biosynthesis. 3.1.7. S-Methylated RiPPs. S-Methylated RiPPs are quite rare; to the best of our knowledge, S-methylations have only been observed in select members of the thiopeptide natural products (Scheme 12).85−87 In each of these cases, the biosynthetic gene clusters, and thus the enzymes required for C−S bond formation, are unknown. Recently, a member of the proteusin RiPP class was proposed to be S-methylated, and the responsible S-methyltransferase was partially characterized. The proteusins are an emerging class of RiPP natural products grouped by their unusually large leader peptide and the presence of radical SAM enzymes in the biosynthetic gene cluster.22 The only known member of the class, polytheonamide, is produced by an unculturable bacterial symbiont of the marine sponge Theonella swinhoei.88 The genome sequence of the organism, named Candidatus Entotheonella factor, indicates that it has the potential to produce a large number of natural products, including a second proteusin.89 Although the corresponding natural product has not been isolated, the posttranslational modifications installed on the core peptide were partially characterized by coexpressing the precursor peptide (PtyA) and select tailoring genes in Escherichia coli.89,90 When PtyA was coexpressed with a methyltransferase

Scheme 9. S-Methylation of an Anomeric Thiol That Leads to the Thioacetal Residue in Lincomycina

a

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Scheme 11. Model for Brassinin Biosynthesis in B. rapa (L.R., Lossen Rearrangement; GST, Glutathione S-Transferase)

Scheme 12. Structures of Known S-Methylated RiPP Natural Products

the family of glycocins, RiPP natural products that are posttranslationally glycosylated at select serine and cysteine residues.22 All characterized glycocins have antibacterial activities, but the mode of action and the role of the glycosylation in the bioactivity are still under investigation.96 The enzyme responsible for C−S bond formation is a glycosyltransferase 2 (GTase-2) protein family member.92,93 Of the known examples, only the glycosyltransferases involved in the biosynthesis of sublancin and the thurandacins, SunS and ThuS, respectively, have been biochemically characterized.92,96,97 Consistent with characterized GTase-2 family members, SunS and ThuS catalyze the transfer of a nucleotide diphosphate-activated glucose molecule to the precursor peptide (Scheme 13). In both cases glycosylation occurs with inversion of the anomeric center, which suggests an SN2 displacement.94,98 On the basis of the structure of glycocin F and enterocin F4− 9 (a glycocin member containing only O-glycosyl residues), it appears that this mechanism is likely conserved in the glycocin family.99,100 Both SunS and ThuS display a remarkable substrate tolerance in terms of the peptide substrates and the sugar donor cosubstrates that can be transferred.92,94,97 SunS is particularly notable among RiPP biosynthetic enzymes in that it does not require the leader peptide for substrate recognition.97,101 Enzyme reactions carried out with truncated versions of the precursor peptide demonstrated that the enzyme recognizes an α-helix N-terminal to the site of modification; however, the molecular details of this interaction are unknown.97 Moreover, ThuS is not chemoselective and can

homologue (PtyS), the peptide was methylated up to two times.90 The modifications were assigned to the thiol groups of cysteine residues in the core peptide by iodoacetamide labeling and mass spectrometry. The biosynthetic gene cluster also contains a gene encoding a bifunctional lanthipeptide dehydratase/cyclase (PtyM). Coexpression of PtyA and PtyM in E. coli resulted in the installation of up to three lanthionine rings on the core peptide of PtyA.89 Moreover, when PtyA was coexpressed with both PtyA and PtyM, a product bearing three lanthionine rings and a single S-methylated cysteine was produced.90 While these data suggest that the proteusin-like RiPP will be S-methylated, the natural product will need to be isolated before a final verdict can be reached. 3.2. S-Glycosyltransferases

In synthetic chemistry, thioglycosides are well-known as potent glycosylation agents.91 In contrast, natural products bearing Sglycosyl residues are rare, likely because glycosyltransferases preferentially load activated sugars such as UDP-glucose onto O-, N-, and C-nucleophiles.49 Even so, S-glycosylations are involved in the biosynthetic pathways of important biologically active natural products such as S-linked glycopeptides, glucosinolates, and lincosamide antibiotics. 3.2.1. Glycocins. Whereas peptidyl O-glycosylation is a very common posttranslational modification, only five examples of S-glycosylated peptides are known: three isolated from a bacterial source (sublancin, glycocin F, and ASM1) and two generated by the in vitro maturation of a precursor peptide (thurandacins A and B).92−95 All of these examples belong to 5529

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potentially harmful plant metabolites. By screening of a cDNA library of B. napus, the first thiohydroximate S-glycosyltransferase implicated in glucosinolate biosynthesis was discovered. The deduced amino acid sequence shows a highly conserved motif for a glucose-binding domain and is overall highly similar to glucosyltransferases characterized in other species. The heterologously produced enzyme was partly characterized in vitro, inferring UDP-glucose/thiohydroximate S-glucosyltransferase activity by measuring glucose incorporation.104 A functionally related putative thiohydroximate S-glucosyltransferase (UGT74B1) was identified in Arabidopsis thaliana.105 In vitro analyses using recombinant UGT74B1 showed its ability to S-glucosylate phenylacetothiohydroximic to yield the corresponding desulfoglucosinolate (Scheme 14). Kinetic analyses of UGT74B1 with thiohydroximates and the decrease in glucosinolate production in mutants lacking a functional ugt74b1 gene suggested that the enzyme represents a designated S-glucosyltransferase in glucosinolate biosynthesis. Notably, UGT74B1 proved to be a versatile biocatalyst for the chemoenzymatic synthesis of desulfoglycosinolates.106 3.2.3. Lincomycin A and Celesticetin. A unique enzymatic S-glycosylation−transglycosylation sequence takes place in lincomycin biosynthesis. The longstanding mystery as to how the C−S bond is formed in lincomycin A was recently solved.107 A functionally uncharacterized gene, lmbE, with homology to mycothiol (MSH)-S-conjugate amidase was inactivated in the lincomycin biosynthetic gene cluster in Streptomyces lincolensis. The resulting mutant strain produced an α-S-linked MSH−lincomycin conjugate (Scheme 15). Accordingly, reconstitution of LmbE activity in vitro demonstrated that this enzyme catalyzed the removal of the sugar moieties from the MSH−lincomycin conjugate. To identify the enzyme responsible for attaching the MSH moiety to the lincosamide sugar, two additional functionally uncharacterized genes, lmbV and lmbT, with homology to MSH-dependent isomerase and glycosyltransferase genes, respectively, were inactivated. Both mutant strains were unable to produce lincomycin; however, in the lmbV mutant strain, a new analogue was produced that bore a β-linkage between

Scheme 13. Peptide S-Glycosylation in the Biosynthesis of Glycocins, e.g., Sublancin

also catalyze the glycosylation of serine and threonine residues; however, S-glycosylation is faster, potentially due to the increased nucleophilicity of the acceptor.94 Given this high level of promiscuity, glycocin glycosyltransferases represent an attractive target for natural product bioengineering efforts. 3.2.2. Glucosinolates. S-Glycosylation is an essential process in the biosynthesis of glucosinolates, which play an important role in plant defense of crucifers (e.g., cabbage and rape/canola).102 These structurally unusual sulfur compounds are also valued as the precursors of the spicy mustard oil components of wasabi and radish, and of sulforaphane, the cancer-protective agent from broccoli. The thioglycosides are actually only storage forms for chemically reactive defense compounds. Upon wounding or infection of the plant, thioglycosidases (myrosinases) cleave the glycosidic C−S bond, thus liberating aglycones with free thiols that readily undergo Lossen-type rearrangements into the corresponding isothiocyanates (Scheme 14). In oilseed rape (Brassica napus), the glucosinolate-derived thiocarbamate progoitrin has implications for food toxicology because it impairs the production of thyroid hormones.103 Thus, glucosinolate biosynthesis in rape has been a target for molecular breeding approaches to reduce the content of

Scheme 14. General Glucosinolate Biosynthetic Pathway Involving S-Glycosylation and O-Sulfation (by Sulfotransferase, SOT)a

a

The thioglycosidic bond is cleaved by a myrosinase, thus triggering a Lossen rearrangement to afford the isothiocyanate. Structures of representative isothiocyanates from plants are shown. Goitrin is formed from the uncatalyzed cyclization of the corresponding isothiocyanate. 5530

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Scheme 15. S-Glycosylation and Transglycosylation Steps in Lincosamide Biosynthesis

SAM-dependent S-methyltransferase (LmbG; see Scheme 9).76,77 In celesticetin biosynthesis, the LmbF homologue, CcbF, catalyzes the oxidative decarboxylation of the cysteine side chain to afford the aldehyde derivative (Scheme 15). This intermediate is subsequently modified to afford the salicylic ester. It should be noted that the concept of sulfur introduction by a substitution reaction with cysteine or a cysteinyl-bearing larger molecule and its subsequent decomposition to a thiol will be revisited in numerous other pathways, specifically in those employing glutathione S-transferases.

lincomycin and ergothioneine (EGT; Scheme 15). Reconstitution of CcbV (the LmbV homologue from the celesticetin BGC) activity in vitro demonstrated that the enzyme catalyzed the SN2 displacement of EGT with MSH to form the α-linked C−S bond found in lincomycin A. Reconstitution of the GTase, LmbT, demonstrated that the enzyme catalyzed the inverting glycosylation of EGT with GDP-activated lincosamide (Scheme 15). The EGT−lincosamide conjugate is then linked to the propylhygric acid by the action of LmbC, LmbN, and LmbD. While these results demonstrated how the α-linked C−S bond was formed, it did not provide an explanation for the maturation of the deglycosylated MSH−lincomycin analogue into lincomycin A. Reconstitution of LmbF, an aspartate aminotransferase fold type I (AAT_1) superfamily member, demonstrated that these enzymes catalyze the pyridoxal 5′phosphate (PLP)-dependent degradation of the cysteine residue to form desmethyl lincomycin A, the substrate of the

3.3. Glutathione-S-transferases

Glutathione-S-transferases (GSTs) are best known for their involvement in xenobiotic-detoxification processes where they yield water-soluble conjugates.108,109 However, related thioconjugate-forming enzymes have also been implicated in biosynthetic pathways, most notably in the construction of the epidithio bridges of fungal diketopiperazines that may serve 5531

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Scheme 16. Nucleophilic Epoxide Opening and S-Conjugation by a Glutathione S-Transferase Leads to the Slow-Reacting Substance of Anaphylaxis; Downstream Peptidases Convert Leukotriene C4 into Leukotriene E4

Scheme 17. Incorporation of a Dithiol Moiety into the Glutathione Diketopiperazine Core by a Specialized GST

as virulence factors. They have also been implicated in leukotriene, glucosinolate, and allicin biosynthesis, as well as in transient C−S bond formation in E-/Z-isomerizations. 3.3.1. Glutathionyl and Cysteinyl Leukotrienes. The glutathione-detoxification pathway in combination with the

eicosanoid pathway gives rise to a group of sulfur-bearing leukotrienes, LTC4, LTD4, and LTE4 (Scheme 16). These compounds are also known as the “slow-reacting substance of anaphylaxis” (SRS-A), as they represent proinflammatory mediators that are produced during acute asthma attacks as 5532

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Scheme 18. Detailed Mechanism for the Thiol-Liberating C−S Lyase Reaction in the Gliotoxin Pathway

Scheme 19. Model for Glucosinolate Biosynthesis Involving a Glutathione-S-Transferase (GST) (CYP, Cytochrome P450; SOT, Sulfotransferase)

Almost five decades ago, isotopic labeling experiments showed that L-(35S)-cysteine is a sulfur source for sporidesmin biosynthesis in P. chartarum.125 Yet, only recently, the mechanistic details for C−S bond formation in ETPs have been elucidated in A. f umigatus. A nonribosomal peptide synthetase, GliP, assembles the DKP scaffold, which is subsequently hydroxylated by the cytochrome P450 (CYP) monooxygenase GliC at the α-carbon position. According to the current biosynthetic model, dehydration would yield an acyliminium intermediate. This electrophilic intermediate is believed to be the cosubstrate of a specialized GST, GliG, which catalyzes the nucleophilic attack of glutathione (Scheme 17).126,127 To degrade the glutathione backbone and liberate the thiol, three distinct enzymes are required. A γ-glutamyl-transferase (GliK) cleaves the isopeptide bond, releasing pyroglutamic acid in the process. The resulting Cys-Gly dipeptide is hydrolyzed by an unusual metal-dependent dipeptidase (GliJ).128 The final step of the degradation cascade is catalyzed by the PLPdependent C−S lyase GliI (Scheme 18).129 The resulting epidithiol is then oxidized by a flavin adenine dinucleotide (FAD)-dependent oxidase GliT to produce the disulfide bridge.55,56,94 On the basis of sequence homologies, the pathway for the introduction of dithiol residues appears to be highly conserved in ETP biosynthesis. Notably, this strategy of oxygenation followed by nucleophile addition mirrors phase I/II xenobioticdetoxification pathways130 found in diverse organisms and is involved in glucosinolate production in crucifers (see section 3.3.3). Given the widespread nature of this detoxification pathway, it is conceivable that additional secondary metabolites are synthesized in an analogous way. 3.3.3. Glucosinolates. As briefly mentioned in section 3.2.2, glusosinolates (often also referred to as mustard oil

well as after allergen and exercise challenge.110 In particular, cysteinyl leukotriene (LTEA) is one of the most potent bronchoconstrictors known to date.111 The glutathionyl-Sconjugate (LTC4) has been identified as the major trigger of stress-induced oxidative DNA damage.112 LTC4, LTD4, and LTE4 are produced de novo from membrane phospholipids in the context of the leukotriene route of the arachidonic acid cascade.113 The epoxide leukotriene A4, which results from leukotriene lipoxygenation,114 is the precursor of leukotrienes C4 and D4.115 “Leukotriene C4 synthase” was found to be a designated glutathione S-transferase that catalyzes the epoxide ring opening by the nucleophilic glutathione thiol. 113 The glutathionyl adduct is then degraded into leukotriene D4 by means of a γ-glutamyl transferase.116,117 Finally, leukotriene D4 is converted into leukotriene E4 by a dipeptidase (Scheme 16).118 A similar merger of detoxification and biosynthetic enzymes has also taken place in the biosynthetic pathways to ETPs and glucosinolates described below. 3.3.2. Epipolythiodiketopiperazines. Numerous natural products that are characterized by diketopiperazine (DKP) cores and transanullar disulfide bridges constitute the large family of epipolythiodiketopiperazines (ETPs).119 The unusual sulfur functionality of ETPs is mainly responsible for their diverse biological activities, because the dithio group promotes redox cycling, formation of reactive oxygen species, and protein conjugation.120,121 Infamous mycotoxins belonging to the ETPs are found among human, animal, and plant pathogens. For example, gliotoxin is a potential virulence factor of Aspergillus f umigatus,54 sporidesmin from Pithomyces chartarum has been shown to be involved in the development of facial eczema in sheep,122,123 and sirodesmin is a virulence factor of the canola pathogen Leptosphaeria maculans.124 5533

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Scheme 20. Model of Allicin Biosynthesis

transform the oxime hydroxyl into the required leaving group for the aforementioned Lossen rearrangement.149 3.3.4. Allicin/Alliin. An S-glutathionylation−degradation route similar to those observed in the biosyntheses of cysteinyl leukotrienes, ETPs, and glucosinolates has been implicated in the biosynthesis of allicin (diallylthiosulfinate). This sulfurcontaining defense molecule from garlic (Allium sativum) and other Allium species possesses various biological activities and is responsible for the characteristic odor of freshly cut or crushed garlic, which actually indicates an induced plant defense mechanism.150 Damage of the plant tissue activates an enzymatic trigger mechanism, which leads to the formation of allicin from the nonproteinogenic amino acid alliin (S-allyl-Lcysteine sulfoxide).151−153 This C−S cleavage reaction is catalyzed by alliinase, a PLP-dependent lyase, and yields ammonia, pyruvate, and an unstable sulfenic acid that readily forms alliin by dimerization and dehydratation.154 On the basis of precursor feeding, radiolabeling experiments, and isolation of γ-glutamyl-S-allyl cysteine, it was assumed that S-allyl cysteine derives from glutathione S-conjugates.155−157 In addition, labeling experiments with 14C-valine yielded radiolabeled methacrylic acid, a possible source of the allylic residue of alliin and plausible Michael acceptor (Scheme 20).158 Yet, the exact reaction and the involvement of a glutathione Stransferase have remained elusive.159 Recently, enzymes for the cleavage of the glutamyl group of the proposed glutathione-derived intermediate have been identified. Three S-allyl-cysteine-forming γ-glutamyl transpeptidases (AsGGT1−3) with different kinetic properties and subcellular localizations were characterized.160 The next step in the pathway is the stereoselective S-oxygenation of S-allyl cysteine by a FAD-dependent monooxygenase (AsFMO1) to yield alliin.161 The preference of AsFMO1 for S-allyl L-cysteine over γ-glutamyl-S-allyl L-cysteine established the order in which the reactions occur in garlic. 3.3.5. Pseurotin (trans to cis Isomerization). GSTs have also been implicated in the biosynthesis of fungal polyketides that lack a C−S bond. The biosynthesis of the pseurotin class of fungal natural products requires the formation of a high-energy cis double bond between carbons 12 and 13; however, the hybrid polyketide synthase−nonribosomal peptide synthetase involved in pseurotin biosynthesis releases the precursor in the E-conformation.162,163 Through a combination of gene-deletion studies and in vitro enzyme-reconstitution assays, a GST

glycosides) are a hallmark of plants in the order Brassicales and contribute to the pungent taste of cabbages and radishes.102 In the natural environment, these structurally intriguing thioglycosides serve as important defense compounds against herbivore attack and microbial infection. Upon attack of the plant by pathogens, the thioglycoside linkage of the glucosinolates is cleaved by specialized β-thioglucosidases called myrosinases.131 The unstable aglycones undergo various rearrangement reactions. Glucosinolate diversity results from the incorporation of various aliphatic and aromatic amino acids.132 Free amines are uniformly converted into aldoxime moieties by CYP monooxygenases.133−137 After decarboxylation, CYPs transform the aldoximes into nitrile oxides, which may serve as electrophiles for C−S bond formation (Scheme 19).138−142 Whereas these reactive intermediates may react with different thiols in vitro, classical plant-feeding studies suggested that cysteine is the preferred sulfur source in vivo.143 However, three recent lines of evidence indicate that glutathione rather than cysteine is the true sulfur donor. First, mutants lacking glutathione biosynthetic genes were not able to induce indolic glucosinolate production upon fungal or herbivore attack.144,145 Second, two GSTs were found to be coexpressed with glucosinolate biosynthetic genes in Arabidopsis.146 Third, GSH adducts were found in Nicotiana benthaminia expressing glucosinolate pathway genes.147 In analogy to gliotoxin biosynthesis, a γ-glutamyl peptidase (GGP1) was identified that could catalyze the first cleavage step to degrade the GSH backbone (Scheme 19). An Arabidopsis double mutant lacking ggp1 as well as a redundant gene ggp3 showed reduced levels of glucosinolates.148 The next known step in the pathway is catalyzed by the C−S lyase SUR1, which has been shown to be essential for glucosinolate production in Arabidopsis.84 SUR1 was recently reconstituted in vitro and was shown to cleave S-cysteine thiohydroximate to thiohydroximate.83 Although the enzyme was not tested for the ability to process the Cys-Gly dipeptide linked aldoxime, these data strongly suggest that the glycine residue is cleaved off prior to SUR1 processing. As outlined in section 3.2.2, the SUR1 reaction product is S-glycosylated by specialized glucosyltransferases to produce desulfoglucosinolates.105 The last step in glucosinolate biosynthesis is catalyzed by 3′-phosphoadenosine5′-phosphosulfate (PAPS)-dependent sulfotransferases, which 5534

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Scheme 21. Glutathione-S-Transferase Mediates E/Z-Isomerization of a Double Bond in Pseurotin Biosynthesis; GSH, Glutathione

3.4. S-Transferases Involved in Conjugate Additions

(PsoE) and a bifunctional methyltransferase/FAD-dependent monooxygenase (PsoF) were shown to be required for the olefin isomerization.164,165 When the 12,13-E-configured intermediate, presynerazol, was incubated with glutathione and NADPH in the presence of PsoE and PsoF, the 12,13-Zconfigured compound synerazol was obtained.164 Importantly, neither enzyme was capable of catalyzing the isomerization alone. The isomerization is proposed to proceed through a Sconfigured glutathione−presynerazol conjugate at C-13 that is subsequently oxidized to the sulfoxide by PsoF (Scheme 21). Although these products have not been isolated, compounds with a mass/charge ratio consistent with these conjugates were detected in the enzyme reactions. The sulfoxide intermediate is thought to undergo a PsoF-catalyzed pericyclic syn-elimination to yield Z-configured presynerazol, which is further oxidized to synerazol by PsoF. The crystal structure of PsoE with bound presynerazol and GSH (Figure 4) shows the characteristic GST

In several biosynthetic pathways, enzymes or domains catalyze the formation of C−S bonds by conjugate addition of a sulfur nucleophile to enones or α,β-unsaturated acids and thioesters. Yet, these biocatalysts are only distantly related to wellcharacterized enzymes such as GSTs and/or use different substrates. In contrast to the cyclases outlined below (section 4.1), which also mediate conjugate additions, the S-transferases included in this section do not lead to intramolecular cyclizations. Notably, mechanistically related reactions can also take place without enzyme catalysis (section 8.1). 3.4.1. Tropodithietic Acid and Roseobacticide A. A GST homologue (TdaB) has been implicated in the biosynthesis of unusual sulfur-substituted tropolone derivatives that regulate the symbiotic interaction of marine alpha-proteobacteria (clade Roseobacter) with their algal hosts. As long as the microalgae (Emiliania huxleyi) provide the bacteria with food, the bacteria (Phaeobacter inhibens) produce tropodithietic acid (TDA), an antibiotic that protects the host. However, the algae release p-coumaric acid upon senescence, which triggers P. inhibens to synthesize algicidal roseobacticides.167 Stable isotope-labeling studies indicated that the carbon backbone TDA is derived from phenylacetic acid and that sulfur amino acid metabolism serves as the source for the sulfur.168,169 On the basis of in silico analyses of the TDA biosynthetic gene cluster, isotope labeling, and mutational studies, a plausible sulfurization pathway was proposed (Scheme 22).170 First, phenylacetic acid is transformed into a seven-membered carbacycle by oxidative ring cleavage. Then, dehydrogenation (by TdaE) and dehydration (by TdaC) yield the tropolone ring. The GST homologue TdaB is believed to catalyze the conjugate addition of S-thiocysteine, rather than glutathione, to nascent tropolone ring. Subsequently, the flavoprotein TdaF could mediate an oxidative cleavage of the disulfide adduct. The thioaldehyde byproduct was proposed to be converted to cysteamine and subsequently recycled in primary metabolism;170 however, it could also spontaneously degrade to hydroxypyruvate, ammonia, and hydrogen sulfide. The reaction cycle is repeated at the adjacent carbon to afford the dithiol. The vicinal thiol groups could undergo either spontaneous oxidation by molecular oxygen or enzymatic-catalyzed oxidation, as in the gliotoxin and holomycin pathways. However, a homologue of GliT or HlmI is not encoded in the TDA gene cluster.

Figure 4. Crystal structure of PsoE in complex with presynerazol− glutathione conjugate (PDB entry code: 5F8B). A zoomed-in view of the active site shows that the conjugate is bound in a shallow, solventexposed pocket, which may promote transfer to PsoF.

fold, yet the sequence similarity of PsoE to other GSTs is low. PsoE has specific structural characteristics that might favor a fast release of the glutathione conjugate and modification by PsoF. A similar GST-mediated isomerization has been implicated in the biosynthesis of hypothemycin; however, the GST in this case is only able to produce a mixture of 85% E and 15% Z products.166 This low level of conversion suggests that an additional enzyme may be required to catalyze the isomerization effectively. To date, an auxiliary enzyme has not been identified and mechanistic insights into this reaction remain missing. 5535

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Scheme 22. Model for the Biosynthesis of Tropodithietic Acid and Roseobacticide Involving a GST-Like Enzyme That Transfers S-Thiocysteine; Box: Biosynthetic Origin of Sulfur and Formation of S-Thiocysteine by PLP-Dependent Degradation of L-Cystine

Scheme 23. Selected Structures of Carbapenem Antibiotics and Model for the Biosynthesis of the Carbapenem Core

been shown to be a source of sulfur that is incorporated into TDA and roseobacticide via cysteine (Scheme 22). The oxidized, dimerized form, cystine, is the substrate for a PLPdependent C−S lyase (PatB), which cleaves the molecule into ammonia, pyruvate, and S-thiocysteine.170,172 This process is analogous to the cysteinyl-degradation pathway studied in gliotoxin biosynthesis (see section 3.3.2). 3.4.2. Thienamycin. Another complex scenario has been observed for the thiolation of the carbapenem core structure en route to thienamycin and related peptidoglycan-targeting

In contrast to TDA formation, only a single TdaB/TdaF reaction cycle is required for roseobacticide biosynthesis. The monothiol intermediate could then be fused to a derivative of p-coumarate, which is released by the senescing algal host, and further modified to afford the algicidal agent (Scheme 22).171,172 The S-methyltransferase responsible for forming the second C−S bond in roseobacticide biosynthesis has not been identified. It is noteworthy that the microalga provides the bacteria food in the form of dimethylsulfoniopropionate (DMSP), which has 5536

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Scheme 24. Two Proposed Routes for the Origin of the Cysteamine Side Chain of Thienamycin; Box: Current Proposals for C− S Bond Formation

antibiotics (Scheme 23). The Streptomyces cattleya metabolite thienamycinthe first carbapenem isolatedinspired the development of many potent members of this family of antibacterial agents.173,174 It should be highlighted that, among the β-lactams currently in clinical use, the carbapenems stand out because they are relatively resistant to hydrolysis by most βlactamases.174 Early stable isotope-labeling experiments indicated that the biosynthesis of thienamycinas all carbapenemsdiffers markedly from the assembly of the structurally related β-lactam ring systems of penicillins and cephalosporins.175 Biochemical analyses showed that the carbapenem scaffold is assembled by a crotonase-like carboxymethylproline synthase (CarB/ThnE) that fuses glutamate semialdehyde (or its cyclized form) and malonyl-CoA (Scheme 23).176−179 The resulting proline derivative is then cyclized into the β-lactam by an ATP-dependent enzyme (CarA/ThnM).178−180 In the case of simple carbapenem antibiotics, the carbapenem ring is epimerized and desaturated by a 2-oxoglutarate-dependent nonheme iron oxygenase (CarC) to give carbapenem carboxylic acid.179,181,182 For complex carbapenems, such as thienamycin, the remaining biosynthetic steps are largely unknown; however, significant progress has been made in recent years. The ethyl side chain at position C2 is introduced by two successive SAM-dependent methylations catalyzed by a cobalamin-dependent radical SAM enzyme (ThnK)175,183,184 and is subsequently oxidized by a nonheme iron-dependent oxygenase (ThnG).185,186 Various late-stage enzymatic diversification steps have been identified, including S-oxygenation and N-acetylation.185−187 However, both the source of the

cysteaminyl side chain and the mechanism of C−S bond formation remain unclear. There are currently two hypotheses regarding the source of the sulfhydryl side chain of thienamycin. Radioisotope-labeling experiments conducted with 35S-cystine and 35S-pantethine demonstrated that, while both could be incorporated into thienamycin, cystine incorporation was significantly more efficient.175 This led to the proposal that the cysteaminyl side chain originated from the conjugate addition and decarboxylation of cysteine. However, the discovery of the OA-6129 series of carbapenems (Scheme 24) in a different Streptomyces sp.188 suggested that the sulfhydryl compound used could be pantetheine. Moreover, a randomly generated mutant of the epithienamycin producer, Streptomyces f ulvoviridis, lost the ability to produce the N-acetylcysteamine-substituted carbapenems and instead formed OA-6129 carbapenems.189 This mutant lacked A933 acylase, which, following purification from S. f ulvoviridis, was shown to cleave the pantetheinyl side chain of OA-6129 carbapenems to afford the cysteaminyl derivatives.190−192 Taken together, these findings suggested that OA6129 group carbapenems are the precursors of the cysteaminesubstituted congeners. Subsequently, in vitro studies demonstrated that three enzymes encoded in the thienamycin biosynthetic gene cluster catalyze the stepwise degradation of coenzyme A into cysteamine.187 It was shown that ThnR (a member of the Nudix hydrolase family) and ThnH (another hydrolase) successively cleave coenzyme A to give pantetheine, and that ThnT hydrolyzes the amide bond between β-alanine and cysteamine (Scheme 24).187,193,194 While these experiments provide support for pantetheine being the sulfur source, insertional inactivation of thnR and thnT did not affect 5537

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Scheme 25. Model of Online Thiol Conjugate Addition and C−S Lyase Reaction on PKS-Bound Intermediates in the Biosynthesis of Leinamycin (DUF, Domain of Unknown Function; SH, C−S Lyase Domain)

is needed to solve the riddle of C−S bond formation in thienamycin and other thioether-containing carbapenems. 3.4.3. Leinamycin. Another obscure conjugate addition of a sulfur nucleophile takes place in leinamycin biosynthesis in Streptomyces atroolivaceus S-140. Leinamycin (LNM) is a sulfurcontaining cytotoxin that is characterized by an unusual 1,3dioxo-1,2-dithiolane moiety spiro-fused to a thiazole-containing 18-membered lactam ring (Scheme 25).199 The 1,3-dioxo-1,2dithiolane moiety is essential for the antitumor activity of leinamycin, as it alkylates DNA through an episulfonium ion intermediate.200,201 The LMN biosynthetic gene cluster has been identified,202−204 and major parts of the NRPS−PKS pathway to the 18-membered macrolactam have been elucidated. Structures of intermediates and shunt products from mutant strains suggested that sulfur is introduced during polyketide chain elongation, yet after β-alkylation at C-3.205−209 In silico analyses identified two uncommon domains within PKS module 8, a domain of unknown function (DUF) and a tentative lyase (SH) domain.210 It was proposed that the DUF could catalyze the conjugate addition of a sulfur nucleophile (e.g., L-cysteine) to an α,β-unsaturated linear polyketide intermediate. This reaction would be analogous to the recently studied online Michael additions of carbon and oxygen nucleophiles to ACP-bound, α,β-unsaturated thioester intermediates.211 Whereas designated PKS domains have been shown to introduce alkyl branches212−214 and pyran rings215−217 by conjugate addition, biochemical support for the proposed vinylogous S-transfer by the DUF domain is missing. However, using L-cysteine and Smodified L-cysteine analogues as in vitro substrates, the SH domain was shown to act as a PLP-dependent C−S bond lyase that catalyzes the β-elimination of the L-cysteinyl residue to give pyruvate, ammonia, and the corresponding thiol group (Scheme 25).210 It should be noted that the still-enigmatic second C−S bond formation is not essential for bioactivity. The thiol-substituted macrolactam (leinamycin E1), the immediate

theinamycin production under standard cultivation conditions.195 Since the sequence of the thienamycin biosynthetic gene cluster was determined in 2003,196 there have been multiple proposals regarding the installation of the C2 side chain onto carbapenem-3-carboxylic acid (Scheme 24). Initially it was thought that the carbapenem ring would be oxidized to the corresponding carbapenem derivative, which could serve as a substrate for the conjugate addition of the thiol. This conjugate addition was thought to be catalyzed by a glutathione-Stransferase homologue (ThnV) encoded in the gene cluster.196 However, thnV homologues are not found in the biosynthetic gene clusters of all sulfur-containing carbapenem antibiotics.186,197 Mutational analysis of the thn gene locus in S. cattleya revealed that, among other genes, thnN and thnO are essential for thienamycin biosynthesis.196 Crossfeeding experiments indicated that ThnN (and likely also ThnO) perform reactions before methylation of the carbapenem ring, yet after the bicyclic core has been produced.195 Thus, it was hypothesized that these two enzymes may be involved in the conjugate addition. Yet, homologues of ThnN, which contains an amino acid adenylation domain, and ThnO, likely a member of the NAD(P)-dependent aldehyde dehydrogenase superfamily, were found to be responsible for the reduction of a carboxylic acid to an aldehyde in the grixazone biosynthetic pathway (see section 6.4.2), casting doubt on this model.198 A third model for C−S bond formation implicates two additional cobalamin-dependent radical SAM enzymes encoded in the biosynthetic gene cluster (ThnL and ThnP).184 In this proposal, sulfur addition happens prior to oxidation of the carbapenem ring and occurs through a radical mechanism. Consistent with this hypothesis, mutant strains lacking thnL and thnP are unable to produce thienamycin and appear to be blocked early in the biosynthetic pathway.195,196 Moreover, the thnP mutant accumulates carbapenem-3-carboxylic acid.195 Thus, while there have been intriguing clues, further research 5538

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Scheme 26. Lanthionine, Labionine, and Aminovinyl Cysteine Ring Formation by Intramolecular Conjugate Addition of a Cysteine Thiol

eCys), and labionin (Lab) (Scheme 26).22,219,220 The biosynthesis of each of these rings begins with the dehydration of individual serine and threonine residues on the precursor peptide by the action of the lanthipeptide dehydratase. To date, three classes of lanthipeptide dehydratases have been characterized. While the classes are not homologous, all three catalyze the activation of the side-chain oxygen followed by elimination of the activated species (Scheme 26). The resultant dehydroalanine (Dha, from serine) and dehydrobutyrine (Dhb, from threonine) residues are further modified by the lanthipeptide cyclase to generate the thioether linkages. For Lan, MeLan, and Lab rings, a cysteine thiol is appended to select Dha and Dhb residues in a Michael-like addition (Scheme 26). Avi(Me)Cys rings are formed in an analogous fashion from the addition of an enethiolate, which is generated from the oxidative decarboxylation of a C-terminal cysteine residue.221−223 There are two classes of lanthipeptide cyclases that are evolutionarily related but are differentiated by the presence or absence of a Zn metal center. Zn-dependent lanthipeptide

product of the lmn assembly line, actually serves as a prodrug that can be activated by reactive oxygen species.218

4. C−S BOND-FORMING CYCLASES To date, two classes of C−S bond-forming enzymes that catalyze intramolecular cyclizations in an ATP- and oxidationindependent fashion have been identified. These cyclases are responsible for the formation of thiazoline rings on NRPS assembly lines and the conjugate additions that form the eponymous lanthionine rings in lanthipeptide biosynthesis. In both cases the cyclizations are important for biological activity because they confer rigidity to the peptide natural products and, in select cases, lead to installation of pharmacophoric groups (e.g., the installation of DNA-intercalating bisthiazoles). 4.1. Lanthipeptide Cyclases

The lanthipeptides are a diverse RiPP subclass containing various S-heterocycles that include the nonproteinogenic amino acids lanthionine (Lan), methyl lanthionine (MeLan), aminovinyl cysteine (AviCys), aminovinylmethyl cysteine (AviM5539

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cyclases ligate a Zn2+ ion with a conserved cysteine−cysteine− histidine/cysteine triad.224−227 The cysteine thiol of the precursor peptide is proposed to ligate the Zn, thereby activating the thiol for the subsequent Michael-type addition. A conserved histidine and aspartate located in the active site are thought to perform the requisite acid/base chemistry during thioether bridge formation.225,228 In contrast, the mechanism of Zn-independent lanthipeptide cyclases is largely uncharacterized. Biochemical experiments demonstrated that the enzymes do not require metals for catalysis suggesting that the enzymes perform thioether formation with acid/base catalysis.229,230 After nucleophilic attack, the enolate intermediate can be resolved in one of two ways. In the majority of lanthipeptides, the enolate is protonated to form the Lan/MeLan/Avi(Me)Cys macrocycle (Scheme 26). However, the enolate intermediate can also catalyze a second Michael-like addition, which, after protonation, results in a Lab macrocycle.229−231 While it is not clear how a subset of lanthipeptide cyclases form Lab rings, it is noteworthy that characterized Lab-forming enzymes are exclusively zinc-independent cyclases. Many lanthipeptides contain multiple Dha/Dhb residues and have multiple macrocycles. While it was originally thought that the regioselectivity of the lanthipeptide cyclase governed the final ring topology of the natural product, work with two divergent lanthipeptide cyclases demonstrated that the naturally occurring macrocycles are formed independent of the cyclase used.232 Moreover, it was demonstrated that the configuration of the thioether ring is governed by the precursor peptide sequence surrounding the acceptor Dha or Dhb.233 Together these results have led to a new model for lanthipeptide cyclization where the regio- and stereoselectivity of the transformation is dictated by the sequence of the precursor peptide rather than by the cyclase. Notably, a similar mechanism is thought to be involved in the sactipeptide biosynthesis (see section 7.1.2) and might be a more general trend in RiPP biosynthesis.234−236 For further reading on the biosynthesis and properties of lanthipeptides, we refer you to recent reviews.219,220,237 In addition to the lanthipeptides, AviCys rings are also found in the linaridin class of RiPP natural products (Scheme 27).22,238 While linaridins were originally thought to be a subclass of lanthipeptide natural products because of the presence of Dha and Dhb residues in addition to the AviCys ring, the identification of the biosynthetic gene cluster for the founding member of the family, cypemycin, demonstrated that neither a lanthipeptide dehydratase nor a lanthipeptide cyclase is involved in biosynthesis.238 Moreover, the precursor peptide sequence demonstrated that the acceptor residue for thioenolate attack is a cysteine in cypemycin instead of a serine as found in AviCys-containing lanthipeptides. Although mutagenesis studies have identified enzymes important for the biosynthesis of linaridins, the enzyme responsible for catalyzing thioether bond formation remains unknown.

Scheme 27. Model for the Biosynthesis of Cypemycin, a Linaridin-Type RiPP

respectively,242,243 the cytotoxic metalloantibiotic bleomycin from Streptomyces verticillus,244,245 and the antimitotic epothilone from Sorangium cellulosum (Scheme 28).246 The biosynthesis of S- and O-heterocycles in nonribosomal peptides has been reviewed extensively.239,241,247 In brief, the formation of the five-membered heterocycles requires the enzyme-mediated cyclocondensation of X-Cys dipeptide residues, catalyzed by Cy (cyclization) or HC (heterocyclization) domains. While these domains are typically embedded in the NRPS module, an example of a trans-acting Cy domain is known.248 To date, multiple Cy domains have been biochemically characterized including, but not limited to, those involved in yersiniabactin, pyochelin, and epothilone biosynthesis.249−251 These studies demonstrated that Cy domains are bifunctional enzymes that catalyze both the condensation reaction and heterocycle formation. As uncyclized intermediates have been observed in reactions performed with Cy domain mutants and substrate derivatives, amide formation is thought to occur first through a mechanism identical to canonical condensation domains (Scheme 28).252−255 Following condensation, the thiazoline heterocycle is formed from the nucleophilic attack of the side chain and subsequent dehydration of the tetrahedral intermediate. NRPS Cy domains have a conserved DxxxxDxxS motif that was originally predicted to be the active site of the protein; however, two recent crystal structures of Cy domains demonstrate that these residues play a structural role (Figure 5).256,257 Instead, an Asp-Thr dyad was implicated in catalysis and shown to be highly conserved in diverse Cy domains. In a subset of biosynthetic clusters, the nascent thiazoline ring is either oxidized to a thiazole or reduced to a thiazolidine by an FMN-dependent oxidase (Ox) or NADPH-dependent reductase (R) domain, respectively (Scheme 28).258−260 As with the Cy domains, Ox and R domains can be encoded within the NRPS module or be stand-alone enzymes. Beyond thiazoline formation, an NRPS Cy domain has been implicated in the ring opening of a thiirane intermediate to form the thiotetronate system of thiolactomycin (see section

4.2. NRPS Heterocyclization Domains

Thiazolidines, thiazolines, and thiazoles are important moieties in many nonribosomal peptide natural products. In addition to increasing the proteolytic stability of the peptide, these sulfurcontaining heterocycles are often critical for the biological activity of the compounds (e.g., metal binding and target recognition).239−241 Notable examples of S-heterocyclic nonribosomal peptides are the siderophores yersiniabactin and pyochelin from Yersinia pestis and Pseudomonas spp., 5540

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Scheme 28. Structures of Select Natural Products Bearing Thiazolidine, Thiazoline, and Thiazole Rings and General Mechanisms for Heterocyclizations

Figure 5. Crystal structures of a NRPS condensation domain (left; PDB entry code: 1L5A) and Cy domain (right; PDB entry code: 5T7Z). The active site HHxxxD motif of the condensation domains is replaced by a structural DxxxxDxxS motif in Cy domains (colored cyan). The putative AspThr catalytic dyad in the Cy domain is indicated (colored green).

5.2.2)yet another avenue to form a thioester moiety (Scheme 29).261 The Cy domain has the typical DxxxxDxxS structural motif of the heterocyclization domains involved in thiazoline/ oxazoline formation. However, mechanistic details for the

thiolactonization with concomitant shifting of the double bond

need to be clarified. 5541

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and Archaea.262 The family is named after the Escherichia coli homologue, which has been implicated in the thiomethylation of the S12 protein of the ribosome.263 For over two decades YcaO protein family members have been known to be involved in the biosynthesis of azol(in)e-containing RiPPs; however, they were thought to play a regulatory role.264 Recently, it was demonstrated that YcaO proteins catalyze a range of ATPdependent cyclodehydrations in RiPPs and may promote other reactions (e.g., thioamide formation). 5.1.1. Thiazoles and Thiazolines in RiPPs. As in NRPSderived peptides, thiazole and thiazoline heterocycles are also found in many ribosomally produced natural products, mainly in members of the cyanobactin (e.g., patellamide), thiopeptide (e.g., thiocillin), linear azol(in)e-containing peptides (e.g., plantazolicin), and bottromycin (e.g., bottromycin A2) subclasses of RiPPs (Scheme 30).22 Although the enzymes responsible for azol(in)e heterocycle biosynthesis have not been characterized in all instances, current evidence suggests a universal strategy for C−S bond formation catalyzed by a cyclodehydratase belonging to the YcaO superfamily of proteins.265,266 This enzyme is not chemoselective and is also responsible for cyclizing serine and threonine residues to generate the (methyl)oxazoline moieties also commonly found in these natural product classes. Isotope-labeling experiments demonstrated that the YcaO cyclodehydratase catalyzes the nucleophilic addition of a cysteine side-chain thiol and the subsequent ATP-dependent activation of the amide carbonyl

Scheme 29. Model for the Thiirane Ring-Opening Reaction Mediated by a Cyclase (Cy) Domain in Thiolactomycin Biosynthesis

5. ATP-DEPENDENT C−S BOND-FORMING ENZYMES In many biosynthetic pathways that do not involve substitution reactions with good leaving groups, the formation of covalent C−S bonds may require ATP. In most cases, ATP is essential to activate a carbonyl and transform an intermediary hydroxyl into a leaving group. However, the classes of the enzymes, the types of sulfur nucleophiles involved, and the resulting functionalities are highly diverse. 5.1. YcaO-like enzymes

YcaO proteins, also known as DUF181 proteins and formerly annotated as docking proteins, are widely dispersed in Bacteria

Scheme 30. Examples of Thiazol(in)e-Containing RiPPs and Model of Heterocycle Biosynthesis

5542

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oxygen (Scheme 30).265−267 Subsequently, the tetrahedral intermediate is resolved by elimination of the carbonyl oxygen as either phosphate or adenosine monophosphate, depending on the specific enzyme, to afford the thiazoline residue.265,267,268 Similar to the biosynthesis of NRPS-derived natural products, the thiazoline heterocycle is further oxidized to yield the thiazole by either a flavin mononucleotide-dependent dehydrogenase or a CYP-dependent decarboxylase (Scheme 30).22,264,269 In all cases where the enzymatic activity has been reconstituted in vitro, a third protein is required for efficient azol(in)e biogenesis. This third protein is responsible for binding the leader peptide region of the precursor peptide, presenting the peptide substrate to the cyclodehydratase and the dehydrogenase, and potentiating the activity of the cyclodehydratase.267,270−272 As is common for RiPP maturation proteins, characterized cyclodehydratases are remarkably promiscuous and are able to accept diverse peptide substrates.264,273 5.1.2. Thioviridamide. In addition to genetic loci coding for azol(in)e-containing RiPPs, a gene encoding a YcaO protein family member was found in the thioviridamide biosynthetic gene cluster.274 Thioviridamide is an unusual natural product in that it contains thioamide linkages (Scheme 31).275 In analogy to thioamide formation in 6-thioguanine biosynthesis (see section 5.2.1), it is thought that the thioamides of thioviridamide are formed through the activation of the carbonyl oxygen and subsequent nucleophilic attack of sulfide. Given the mechanistic similarities to thiazoline formation in RiPPs, the YcaO protein encoded in the thioviridamide gene cluster has been proposed to perform this transformation (Scheme 31).272,274 However, further research will be needed to determine if this biosynthetic proposal is correct.

member. As members of this family are known to catalyze C−S bond formation in thionucleoside biosynthesis, YcfA was proposed to carry out thioamide formation in 6TG biosynthesis. On the basis of the model of the catalytic cycle of the 2thiouridylase MnmA and the 4-thiouridylase ThiI,7,283,284 YcfA is thought to catalyze the adenylation of guanine followed by the nucleophilic addition of sulfide and elimination of carbonyl oxygen as AMP (Scheme 32).281 The highly reactive persulfide Scheme 31. Model of Thioamide Formation Involving a YcaO-like Enzyme in Thioviridamide Biosynthesis

moiety required for YcfA-mediated thiolation is likely provided by a sulfur-relay system that includes a cysteine desulfurylase and sulfur carrier proteins. Mutational studies of the three cysteines in YcfA indicated that only Cys113 was required for 6TG formation when the cluster was expressed in E. coli. It is thought that this cysteine accepts sulfide from the sulfur-relay system as a cysteine persulfide and is responsible for sulfur transfer to the adenylated nucleobase. After C−S bond formation, AMP elimination would afford 6TG. It is noteworthy that, in thiouridine formation, sulfur release from the persulfide requires the formation of an active site disulfide with a second cysteine residue.283,284 This disulfide is subsequently reduced to regenerate the active enzyme. As only a single cysteine residue has been shown to be required for YcfA activity, it is unclear how this AANH-like family member carries out the critical C−S bond-forming step. 5.2.2. Thiolactomycin. Surprisingly, a YcfA homologue catalyzes a key step for introducing sulfur into thiolactomycinbut by a strikingly different mechanism and a highly specialized sulfur-relay system. Thiolactomycin is a bacterial thiotetronate antibiotic that selectively inhibits type II fatty acid synthases.285−287 Radioisotope-labeling experiments using 35SL-cysteine in a Nocardia sp. showed that the sulfur atom of thiolactomycin derives from L-cysteine, likely by a process similar to the IscS-catalyzed sulfur relay in E. coli. A biosynthetic model involving a thiirane intermediate was proposed that could explain thiolactone formation with concomitant double-bond shift.288 The recent identification of tlm biosynthetic gene clusters in Streptomyces thiolactonus and Salinispora pacif ica confirmed this proposal and shed more light on the details of thiolactomycin assembly.261,289 The backbone of the polyketide is assembled by an iterative PKS−NRPS.261,289,290 Mutational analyses

5.2. Adenine Nucleotide Alpha Hydrolase Enzymes

Adenine nucleotide alpha hydrolase enzymes catalyze critical transformations in a diverse set of primary metabolic pathways. Select members of the protein family catalyze sulfur introduction into nucleobases as a posttranscriptional RNA modification.276,277 These thiolation reactions are ATP-dependent and proceed through a complex sulfur-relay system. Interestingly, similar strategies involving thiouridylase-like enzymes and sulfur relay systems have been identified in two natural product biosynthetic pathways. 5.2.1. 6-Thioguanine. The first enzyme responsible for thioamide formation in a natural product was identified in the context of 6-thioguanine biosynthesis. 6-Thioguanine (6TG) is a thiopurine in clinical use as an anticancer agent.278,279 Although 6TG has been known as a synthetic antimetabolite since the 1950s, it was not until the late 1980s that the compound was isolated from a natural source, the plant pathogen Erwinia amylovora.280 E. amylovora is the causative agent of fire blight, a devastating disease affecting trees in the Rosaceae family (mainly apples and pears). Only recently, it was shown that 6TG plays a major role as an E. amylovora virulence factor in fire blight.281 Gene inactivation in E. amylovora and heterologous expression in E. coli revealed that 6TG biosynthesis is encoded by the “yellow compound formation” (ycf) gene cluster and that four genes (ycfABCD) are responsible for 6TG biosynthesis.281,282 Through bioinformatics and three-dimensional structure predictions, YcfA was identified as an adenine nucleotide alpha hydrolase-like (AANH-like) superfamily 5543

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Scheme 32. Thioamide Formation in 6-Thioguanine Biosynthesis Parallels Thiouridine Formation

revealed the essential role of a CYP monooxygenase (TlmD1) in TLM biosynthesis.261 According to the current model, TlmD1 would epoxidize the ACP-bound full-length polyketide. Then, an ATP-dependent thiouridylase (TlmJ) would deliver a persulfide that converts the epoxide into the postulated thiirane intermediate. Analogous to the mechanism of the thiouridylase enzymes and proposed mechanism for YcfA, TlmJ is thought to catalyze the adenylation of the hydroxyl group to facilitate thiirane formation (Scheme 33). The persulfide group in TlmJ is likely regenerated by the pathway-specific cysteine desulfurase TlmS.261 The cysteine substrate of TlmS is likely activated by the adenylation (A) domain and tethered to the PCP domain of the PKS−NRPS enzyme, which would allow a fast and continuous supply of activated sulfur. After sulfur transfer, the adjacent thioesterase domain could cleave PCP-bound alanine, regenerating the PCP for a new reaction cycle (Scheme 33). The final step, the cyclization into the thiotetronate system, is likely catalyzed by the cyclization (Cy) domain (see section 4.2). It is noteworthy that this peculiar thiotetronate biosynthetic pathway is encoded in various bacteria across distinct genera, including marine bacteria.261,289

Scheme 33. Model for the Biosynthesis of Thiolactomycin Involving a Thiirane Intermediate

5.3. Adenylating Protein/Sulfur Carrier Protein Systems

In primary metabolism, sulfur is often transferred via thiocarboxy adenylate sulfur carrier protein systems, in which a carboxy residue is activated by ATP and transformed into the corresponding thiocarboxy group by attack of a persulfide.7,291,292 The source of the persulfide varies but is provided by a cysteine desulfurase in many cases, including the biosynthesis of the important cofactors molybdopterin and thiamine. Remarkably, this strategy of C−S bond formation has been adopted by at least two natural product biosynthetic pathways. 5.3.1. Thioquinolobactin. Thioquinolobactin (TQB) is an unusual Pseudomonas f luorescens metabolite featuring a rare thiocarboxylate residue,293 which is essential for the antifungal activity of the molecule.294 However, the thiocarboxylate moiety is prone to rapid hydrolysis, yielding quinolobactin, an iron chelator.293 The heterocyclic core results from the oxidative cleavage of the tryptophan indole and cyclization of the intermediary hydroxykynurenine.295 Although the mechanism of C−S bond formation has not fully been elucidated, incorporation of sulfur has been found to be remarkably similar to mechanisms found in primary metabolism.296 In such pathways, the C-terminus of a small sulfur carrier protein is adenylated by a member of the E1 superfamily. This adenylated intermediate is subsequently transformed into a thiocarboxylate by transfer of sulfur from a cysteine desulfurase bound persulfide.7,291,292 In agreement with this model, the TQB biosynthetic gene cluster in P.

f luorescens codes for a small sulfur carrier protein (QbsE) and a didomain protein composed of an E1 domain and a sulfurtransferring rhodanese domain (QbsC).296 Reconstitution of thiocarboxylate formation in vitro demonstrated that an additional protein, a metallopeptidase (QbsD), is required for activity. This enzyme was shown to cleave off the final two amino acids on the C-terminus of QbsE (-GGCF) to generate the signature digylcine motif that is conserved in the sulfur 5544

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ThiS.6,301−303 As the BE-7585A biosynthetic gene cluster lacks a ThiS homologue, the sulfur carrier proteins from primary metabolic pathways in A. orientalis were screened as sulfur sources for the BexX reaction.304 While ThiS from A. orientalis was not a competent sulfur donor, BexX reactions performed with the thiocarboxylate-bearing sulfur carrier proteins from cysteine (AoCysO) and molybdopterin (AoMoaD2) pathways afforded 2-thioglucose-6-phosphate. Thiocarboxylate formation on AoCysO and AoMoaD2 required the addition of MoeZ, a bifunctional E1 and rhodanese homologue. In analogy to the QbsC reaction in THN biosynthesis, thiosulfate was the sulfur source used in vitro. A mechanism for 2-thiosugar formation has been proposed (Scheme 35). First, a covalent adduct between BexX and G6P is formed that tautomerizes to the 2-keto-bearing sugar. This adduct was directly detected by mass spectrometry and was also present in a cocrystal structure of BexX and G6P (Figure 6).300,304 The C-2 ketone is attacked by the sulfur from the sulfur carrier protein. A cocrystal structure of BexX and AoCysO demonstrates that the C-terminus of the sulfur carrier protein is in close proximity to the G6P binding site (Figure 6). The resultant tetrahedral intermediate is subsequently dehydrated and tautomerized to form the imine. Finally, hydrolysis of the imine linkage releases the product from BexX.299

carrier protein family. Following proteolysis by QbsD, QbsE was transformed into the thiocarboxylate by QbsC using thiosulfate as a sulfur source. In analogy to characterized thiocarboxylate-forming enzymes, a mechanism for the QbsC-catalyzed sulfur transfer was proposed (Scheme 34). The steps leading to sulfur transfer Scheme 34. Model for the Biosynthesis of Thioquinolobactin; Red, Reduction

6. OXYGENASES Oxygenases are often indirectly involved in the formation of C−S bonds, as they set the stage for the attack of a sulfur nucleophile. Thus, as has been outlined above, S-transferring enzymes often come in pairs with oxygenases; for examples, see the biosynthetic pathways of gliotoxin (CYP and GST, section 3.3.2), glucosinolates (CYP and GST, section 3.3.3), and thiolactomycin (CYP and thiouridylase, section 5.2.2). In camalexin biosynthesis, a CYP enzyme generates a reactive intermediate that forms an S-conjugate, yet the potentially involved S-transferase is unknown. In this section a variety of oxygenases are presented that mediate the formation of C−S bonds. In some cases one may question whether these biocatalysts catalyze the covalent linkage of carbon and sulfur atoms or just produce highly reactive compounds that spontaneously react with sulfur-bearing molecules.

from the QbsE-thiocarboxylate to quinolobactin are currently unknown. However, it is thought that quinolobactin is activated by a bifunctional AMP-ligase/methyltransferase (QbsL) and subsequent sulfur transfer is catalyzed by an Acyl-CoA transferase homologue (QbsK). 295 In support of this biosynthetic proposal, homologues of qbsL and qbsK are found in the biosynthetic clusters of two additional thiocarboxylate-containing natural products: the structurally similar compound 2,6-pyridinedicarbothioic acid, produced by Pseudomonas stutzeri, and yatakemycin, produced by Streptomyces sp. TP-A0356.297,298 Moreover, it is noteworthy that the biosynthetic gene cluster of 2,6-pyridinedicarbothioic acid encodes homologues of QbsC, QbsD, and QbsE.298 While the functions of these proteins have not been verified, their presence suggests that this strategy for sulfur mobilization is used in the biosynthesis of additional natural products. 5.3.2. BE-7585A. A related, yet even more complex pathway has been unraveled for the biosynthesis of BE7585A, an antibiotic containing a rare 2-thiosugar. Analysis of BE-7585A biosynthesis in Amycolatopsis orientalis subsp. vinearia implicated BexX, homologue of the thiamine thiazole synthase ThiG, in 2-thiosugar biosynthesis.299 Initial BexX in vitro assays demonstrated that the enzyme could form a covalent adduct with the presumed precursor of the thiosugar, glucose-6-phosphate (G6P); however, the sulfur donor remained enigmatic.300 The mechanism of ThiG has been heavily investigated, and it is known that the sulfur donor for the reaction is the thiocarboxylate of the sulfur carrier protein

6.1. Cytochrome P450 Monooxygenases

Cytochrome P450 monooxygenases are widespread enzymes that are well-known for their ability to transform a broad range of substrates.305 Most commonly, they catalyze hydroxylations and epoxidations, but also more unusual reactions such as sixelectron oxidations of methyl groups into carboxyl groups, aryl couplings, and the formation of nitrile oxide (see section 3.3.3) have been identified. Several CYPs have been implicated in C− S bond-forming reactions leading to the thiazole of camalexin, the S-heterocycles of cyclobrassinin, spirobrassinin, thienodolin, and griseoviridin, and the sulfoxide-substituted ansa-compound ustiloxin. 6.1.1. Camalexin. In addition to glucosinolates, various plants from the crucifer family, e.g., Arabidopsis thaliana, produce camalexin, a thiazole-containing phytoalexin with antifungal activity.306,307 Notably, the route to the thiazole heterocycle markedly differs from the well-known cyclocondensation reactions observed for ribosomal and nonribosomal peptides (see sections 4.2 and 5.1.1). In vitro assays revealed that camalexin biosynthesis in A. thaliana requires the combination of three CYP monoox5545

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Scheme 35. Mechanism for BexX-Catalyzed 2-Thioglucose-6-phosphate Formationa

a

Thiocarboxylate formation on the sulfur carrier protein is shown in the box. Two possibilities for the source of sulfur are indicated. CD, cysteine desulfurase; SCP, sulfur carrier protein

ygenases.308 First, in analogy to glycosinolate pathways, CYP79B2/3 catalyzes the formation of indole-3-acetaldoxime from tryptophan (Scheme 36).309 The oxime is then processed into indole-3-acetonitrile by CYP71A13 or CYP71A12, apparently redundant enzymes, followed by hydroxylation to give a cyanohydrin, in analogy to the biosynthesis of cyanogenic glycosides. In fact, indole-3-carboxyaldehyde has been observed as a side product of cyanhydrin decomposition (Scheme 36). However, in the presence of thiols such as glutathione and cysteine, the enzyme assay gave the corresponding S-adducts. One may conceive that the hydroxylated indole-3-acetonitrile forms an iminium species that could be attacked by sulfur nucleophiles. Although this key step may take place without enzyme catalysis, the slow rate for the formation of the cysteine−indole-3-acetonitrile adduct suggests that a designated transferase (e.g., a GST) may be requiredas in the gliotoxin and glucosinolate biosynthetic pathways. The final oxidative heterocyclization to the thiazole is promoted by CYP71B15. 6.1.2. Cyclobrassinin and Spirobrassinin. The S-heterocycles cyclobrassinin and spirobrassinin from Chinese cabbage (Brassica rapa) are remarkable examples of glucosinolatederived phytoalexins. The dithiocarbamoyl brassinin, which derives from the corresponding isothiocyanate (section 3.1.6), has been identified as the precursor to both cyclobrassinin and

Figure 6. Crystal structures of Amycolatopsis orientalis BexX/CysO complex and BexX covalent intermediate. (A) The BexX/CysO complex (PDB entry code: 4N6E) is displayed. BexX and CysO are colored gray and orange, respectively. A zoomed-in view of the active site is shown to highlight the close proximity of the CysO C-terminus and the lysine responsible for glucose-6-phosphate tethering. (B) A zoomed-in view of the active site of BexX cocrystallized with glucose6-phosphate (PDB entry code: 4N6F) is displayed.

spirobrassinin (Scheme 37).82 RNA sequencing facilitated the identification of upregulated phytoalexin biosynthetic genes in 5546

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Scheme 36. Model for Camalexin Biosynthesisa

a

The C−S bond is also formed nonenzymatically in vitro.

Scheme 37. CYP-Mediated S-Heterocyclizations of Brassinin into Spirobrassinin and Cyclobrassinina

a

Wasalexin is produced from brassinin by an unknown pathway.

Scheme 38. CYP-Mediated S-Heterocyclization in the Thienodolin Pathway

cabbage leaves elicited with Pseudomonas syringae pv maculicola. Various CYP monooxygenase genes were selected and individually expressed in a Saccharomyces cerevisiae strain equipped with the CYP reductase ATR1. Using brassinin as the substrate, the microsomal fraction of CYP71CR1- and CYP71CR2-expressing strains catalyzed the formation of cyclobrassinin and spirobrassininol, respectively. On the basis of labeling experiments, it was concluded that brassinin is epoxidized at the C2,C3 position. Depending on the site of

nucleophilic oxirane ring opening, either a six-membered or a five-membered S-heterocycle is formed, followed by dehydration (likely spontaneous) or CYP-mediated oxidation of the resulting alcohols (Scheme 37).310 Brassinin was also identified as an intermediate for the biosynthesis of wasalexin, a phytoalexin with antifungal activity produced by salt cress (Thellungiella salsuginea) and first isolated from wasabi (Eutrema japonica).311 Although the genes for wasalexin production are unknown, brassinin was 5547

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established as an intermediate by labeling studies and retrobiosynthetic analysis.312 6.1.3. Thienodolin. In a similar way, a CYP may catalyze the formation of thienodolin (THN), a bacterial S-heterocycle that inhibits nitric oxide synthases.313 The gene cluster responsible for the production of THN was recently discovered in the genome of Streptomyces albogriseolus.314 On the basis of gene inactivation experiments and in vitro analyses, a biosynthetic model was proposed that involved the sequential formation of two C−S bonds (Scheme 38). The pathway is initiated by the halogenation of tryptophan.314 Then, aminotransferase ThnJ converts 6-chlorotryptophan into 6-chloroindole-3-pyruvic acid,315 into which sulfur may be introduced by an unknown mechanism, possibly catalyzed by the concerted action of a dehydrogenase, an isopeptidase, and a protein of unknown function encoded in the biosynthetic gene cluster (ThnD−F). The second C−S bond may be formed by a sequence that is analogous to cyclobrassinin biosynthesis.310 The C2−C3 of the indole could be oxidized by a CYP monooxygenase to afford an epoxide, which is opened by nucleophilic attack of the thiol. Subsequently, dehydration and oxidation of the resultant S-containing heterocycle would provide 6-chlorothieno[2,3]indole-2-carboxylic acid (Scheme 38).315 Finally, an amidotransferase (ThnA) introduces the carboxamide function of THN.314 6.1.4. Griseoviridin. Another type of CYP-mediated Sheterocyclization produces the unusual thioene ring of the macrolide griseoviridin, a ribosome-targeting antibiotic from Streptomyces griseoviridis.316 Through analysis of the griseoviridin biosynthetic gene cluster, targeted knockouts, and mutant complementation, it was shown that a CYP monooxygenase (SgvP) is required for the formation of the C−S bond.317,318 The structure of the PKS−NRPS-derived product suggests that the S-heterocycle is in part derived from cysteine. Surprisingly, a mutant lacking SgvP produces a shunt product with a dehydroalanine residue (Scheme 39). Two possible mechanisms for the CYP-mediated cyclization were proposed: a radical mechanism involving a sulfur- and an α-carbon-centered radical and a nucleophilic mechanism invoking an epoxide intermediate (Scheme 39). This latter route resembles the proposed pathways for the brassinin derivatives and thienodolin. 6.1.5. Ustiloxin. A hallmark of the ustiloxins is an unusual aryl sulfoxide linkage formed by the combination of a CYP and a flavoenzyme (Scheme 40). This structurally remarkable group of antimitotic phytotoxins is produced by the causative agent of rice false smut, Ustilaginoidea virens.319−321 A recent report demonstrated that ustiloxins are also produced by Aspergillus f lavus, which facilitated elucidating the molecular basis of their biosynthesis.322 Although the peptide structures of these natural ansa-compounds initially suggested a nonribosomal origin, the discovery of the ustiloxin biosynthetic gene cluster in A. f lavus demonstrated that ustiloxins are members of the RiPP class of natural products.22,323,324 Deletion mutagenesis experiments performed on the ustiloxin biosynthetic gene cluster in A. f lavus demonstrated that the macrocycle is derived from a stretch of four amino acids within a gene-encoded precursor peptide.324 The precursor peptide encodes multiple copies of a small structural peptide that are separated by protease recognition sites.323−325 Proteolytic digestion of the precursor peptide affords the tetrapeptide, which is then posttranslationally modified by eight proteins to give the final natural product ustiloxin B (Scheme 40).324,326

Scheme 39. CYP-Mediated Heterocyclization in Griseoviridin Biosynthesisa

a

Two possible mechanisms for SgvP-catalyzed C−S bond formation are displayed.

The deletion of the ustiloxin maturation enzymes in A. f lavus lead to the production of biosynthetic intermediates that facilitated the characterization of the biosynthetic pathway.326 Deletion of ustC, which encodes a cytochrome P450 family protein, resulted in the production of an intermediate lacking the sulfinyl amino acid on the phenyl ring. Furthermore, the deletion of ustF1, which encodes a flavin-dependent monooxygenase, resulted in a strain producing an intermediate bearing a cysteine moiety on the phenyl ring. The activity of UstF1 was reconstituted in vitro, and the enzyme was shown to oxidize the cysteinyl thioether to the sulfonyl derivative. While the activity of UstC has yet to be reconstituted in vitro, these data suggest that this cytochrome P450 homologue is responsible for C−S bond formation in ustiloxin biosynthesis. Additional support for this functional assignment is provided by the absence of a ustC homologue in the biosynthetic gene clusters of similar fungal RiPPs that lack the arylsulfoxide moiety (e.g., phomopsin, Scheme 40).327 In analogy to the aforementioned examples, UstC likely catalyzes C−S bond formation through an epoxide intermediate (Scheme 40). A similar thioether linkage is formed during the biosynthesis of the potent fungal toxins α-amanitin (Scheme 40) and phalloidin.328 These macrocyclic RiPPs are produced by members of the phylum Basidomycota and harbor a tryptathionine moiety originating from core peptide-encoded Cys and Trp. While the biosynthetic gene clusters for each of these natural products are known and the enzymes responsible for core peptide macrocyclization have been characterized, it is still unclear how the tryptathionine cross-bridge is formed.17,329−331 A ustC homologue is not found in the local 5548

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Scheme 40. Arylsulfoxide Formation in Ustiloxin Biosynthesisa

a

A possible reaction mechanism for the UstC-mediated C−S bond-forming reaction is displayed in the box.

genomic region of the precursor peptide and peptidase in either instance. Further work will be required to identify the mechanism of C−S bond formation in ustiloxin biosynthesis and to identify the enzyme responsible for the analogous modification in α-amanitin and phalloidin maturation.

Scheme 41. General Scheme for the Biosynthesis of Penicillins and Cephalosporins from an NRPS-Derived Tripeptide; ACVS, Aminoadipyl-Cysteinyl-Valine Synthetase

6.2. Nonheme Iron-Dependent Enzymes

Like CYPs, nonheme iron-dependent oxygenases are best known to oxygenate various substrates though the introduction of molecular oxygen. In addition, these versatile enzymes catalyze a broad range of reactions including C−C cleavage and halogenation.332,333 By catalyzing the formation of C−S bonds, nonheme iron-dependent oxygenases also play key roles in the biosynthetic pathways of penicillins, cephalosporin, ergothioneine, and ovothiol. In particular, the detailed analysis of the biosynthesis of penicillins and ergothioneine greatly enhanced our understanding of radical sulfurization reactions. 6.2.1. Penicillins and Cephalosporins. Penicillins and cephalosporins are important β-lactam antibiotics in clinical use. Whereas penicillins are characterized by thiazolidine rings, a dihydrothiazine is typical for the cephalosporins. The Sheterocycles of all naturally occurring penicillins and cephalosporins derive from a single tripeptide, D-(L-α-aminoadipyl)-Lcysteinyl-D-valine, which is assembled by a nonribosomal peptide synthetase (ACVS) (Scheme 41).334−336 In diverse penicillin-producing fungi and bacteria, isopenicillin N synthase (IPNS), a nonheme iron-dependent enzyme, mediates the formation of the canonical penicillin core structure, the fourmembered β-lactam ring fused to the five-membered thiazolidine.335−338 The reaction mechanism of IPNS has been explored in detail through kinetic, spectroscopic, structural, and computational studies.335,339 The combined results from these investigations

were used to formulate a reaction mechanism (Scheme 42). First, the deprotonated thiol group of the tripeptide substrate (ACV) binds to the iron center, which facilitates the binding of molecular oxygen and subsequent formation of a ferricsuperoxo species. The Fe(III)-superoxo complex extracts a hydrogen atom from C-3 of the cysteine, yielding a ferroushydroperoxy intermediate and a thioaldehyde. The valine amide nitrogen is deprotonated by the hydroperoxy moiety, which sets the stage for the formation of the β-lactam ring via nucleophilic attack at the thiocarbonyl. The ferryl-oxo species formed during this process abstracts a hydrogen atom from the β-carbon of valine, and the resulting isopropyl radical reacts with the thiolate sulfur to afford the thiazolidine ring. Recently, additional support for this mechanism was obtained using ACV substrate analogues. The presence of a radical at the β-carbon of valine was probed by use of substrate derivatives in which valine was replaced by amino acids 5549

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Scheme 42. Model for Radical-Based C−S Bond Formation in Penicillins Catalyzed by Isopenicillin N Synthase

Figure 7. Crystal structure of isopenicillin N synthase from Aspergillus nidulans in complex with δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine, iron, and nitric oxide (PDB entry code: 1BLZ). The zoomed-in view shows the active site. Fe-ligating residues are colored cyan.

containing cyclopropylmethyl groups.340 Consistent with the proposed reaction mechanism, radical clock intermediates resulting from the rearrangement of the cyclopropylcarbinyl radical were observed in reactions performed with these substrates. Furthermore, the first spectroscopic evidence for the ferric-superoxo and ferryl-oxo species was obtained using deuterated substrates that displayed significant primary kinetic isotope effects. As previous work had demonstrated that both C−H bond-cleavage events are rate-limiting,339 these substrates were used to accumulate the hydrogen-abstracting species, which was then characterized by Mössbauer spectroscopy.341 The crystal structure of IPNS shows that the iron center is coordinated by two histidine residues and an aspartic acid residue.342 Crystals obtained anaerobically demonstrate that the remaining ligation sites on the iron center are occupied by ACV and two water molecules.343 Although a crystal structure of IPNS bound to AVS and oxygen has not been obtained, a surrogate structure was solved with the oxygen analogue nitric oxide (Figure 7). The nitric oxide binds opposite of the aspartate ligand and immediately adjacent to the substrate. As oxygen would likely bind in a similar manner, this configuration would place the superoxo species in the appropriate position to extract the C-3 hydrogen of cysteine. The cephalosporin β-lactam dihydrothiazine scaffold results from an oxidative expansion of the penicillin ring system, which affords the formation of a new C−S bond (Scheme 43). This committed step in the biosynthesis of all cephalosporin antibiotics is catalyzed by deacetoxycephalosporin C synthase (DAOCS), a 2-oxoglutarate-dependent nonheme iron-depend-

ent enzyme.335 Following ring expansion, the remaining methyl group of deacetoxycephalosporin C (DAOC) is hydroxylated. While this hydroxylation is performed by a second 2oxoglutarate-dependent nonheme iron oxygenase in bacteria (DACS), fungal DAOCS homologues are bifunctional (denoted DAOC/DACS) and catalyze both ring expansion and hydroxylation in a single active site. Interestingly, DAOC/ DACS hydroxylation activity was abolished by the mutation of an active site residue (M306I), converting the bifunctional enzyme into an expandase.344 Early insights into the mechanism of DAOCS were obtained using isotopically labeled substrates. These studies determined the fate of each of the methyl groups of penicillin N and provided evidence of a long-lived radical species during the transformation.345−348 Together with structural and biochemical data,349−354 a reaction mechanism was proposed that follows the consensus mechanism for 2-oxoglutarate-dependent oxygenases (Scheme 43). First 2-oxoglutarate and molecular oxygen react to form a ferryl-oxo species, which extracts a hydrogen from penicillin N. The initial methyl radical is thought to rearrange to an episulfide radical intermediate, which subsequently opens to afford a tertiary carbon radical at C3. The C3 radical is resolved by transfer of a hydrogen radical to the metal center, yielding deacetoxycephalosporin C. Although the initial structural studies suggested that DAOCS operated through a ping-pong mechanism where succinate release would precede penicillin N binding (Figure 8),350,351 recent presteady-state kinetics and NMR-/MS-based binding studies indicate that a ternary (DAOCS·Fe(II)·2-oxoglutarate· 5550

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Scheme 43. Mechanism of DAOCS-Catalyzed Ring Expansion of Penicillin N into Cephalosporin Precursors; 2-OG, 2Oxoglutarate

penicillin N) complex is formed.354 For additional information on the biosynthesis of β-lactam antibiotics, readers are referred to extensive reviews on the topic.335,336 6.2.2. Ergothioneine and Ovothiol. Ergothioneine (EGT) is a histidine derivative with a thiol substituent at the C-2 of the imidazole ring. EGT has been found in a broad range of bacteria, fungi, plants, and animals, where it likely plays a role in cellular redox homeostasis.4,355 Notably, only fungi and bacteria biosynthesize EGT; therefore, other organisms must acquire it through symbioses with producing organisms or through their dietary sources. Humans possess a highly specific transporter (ETT) that promotes the uptake of EGT, which cannot diffuse across the plasma membrane due to its zwitterionic nature.356 The biosynthetic pathway for EGT was identified in Mycobacterium smegmatis by genome mining for a SAMdependent methyltransferase specific to EGT producers and in a local genomic region containing a PLP-dependent decarboxylase.357 Reconstitution of the methyltransferase (EgtD) demonstrated that the enzyme catalyzes the permethylation of the histidine amino group to afford hercynine. Hercynine serves as the substrate for the nonheme iron-dependent enzyme EgtB, which is responsible for C−S bond formation (Scheme 44).357,358 The cosubstrate for the EgtB reaction, γ-glutamyl cysteine, is provided by EgtA, which catalyzes the condensation of glutamate and cysteine.357 The crystal structure of an EgtB homologue from Mycobacterium thermoresistible in complex with manganese, γglutamyl cysteine, and the substrate analogue N-α-dimethyl histidine has been solved.358 The metal center is coordinated by three histidine residues, the imidazole ring of hercynine, the

Figure 8. Crystal structure of cephalosporin synthase from Streptomyces clavuligerus. (Top) Catalytic center of DAOCS in complex with Fe(II) and 2-oxoglutarate (2OG; PDB entry code: 1UOB). (Bottom) Catalytic center of DAOCS in complex with Fe(II) and the substrate penicillin G (penG; PDB entry code: 1UOF). In these structures the binding sites of penicillin G and 2OG overlap. Feligating residues are colored cyan.

5551

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Scheme 44. Biosynthesis of Ergothioneine in Bacteria and Fungi; Box: Product of the EGT C−S Lyasea

a In the absence of a reductant, the hypothesized 2-sulfenic acid intermediate disproportionates to form EGT and EGT-2-sulfinic acid. In the presence of a reductant, EGT is formed directly.

thiol of γ-glutamyl cysteine, and a water molecule in an octahedral geometry (Figure 9). Combined with assays

hydroperoxo species. The thiyl radical could then react with hercynine to form the iminyl radical, which is subsequently rearomatized through deprotonation and a ligand-to-metal electron transfer. Finally, S-sulfoxidation completes the reaction cycle. The thiol residue of ergothioneine is liberated by means of at least two pathway-specific enzymes. First, aminohydrolase EgtC cleaves glutamate from the γ-glutamyl cysteine sulfoxide conjugate (Scheme 44).357,359 Second, a PLP-dependent lyase (EgtE) cleaves the C−S bond of the cysteine-derived moiety, producing pyruvate, ammonia, and EGT (Scheme 44).360 This reaction is reminiscent of the GliI-mediated C−S-cleavage reaction in ETP biosynthesis (see section 3.3.2). When EgtE reactions are performed in the absence of a reductant (e.g., dithiothreitol), the production of EGT-2-sulfinic acid is also observed (Scheme 44). EGT-2-sulfinic acid is proposed to originate from the production of the corresponding sulfenic acid. When a reductant is present, this sulfenic acid intermediate can be reduced nonenzymatically to afford ergothioneine. Notably, the lyase also accepts the corresponding thioether as substrate, thus eliminating the need for a reductant in the transformation. In comparison to the four-step mycobacterial pathway, EGT biosynthesis in fungi is composed of just three steps (Scheme 44).361−365 In characterized fungal pathways, the homologues of EgtD and EgtB are fused in a single protein (Egt1). In vivo and in vitro characterization of Egt1 demonstrated that this

Figure 9. Crystal structure of EgtB from Mycobacterium thermoresistible in complex with γ-glutamyl cysteine (γGC), N-α-dimethyl histidine (DAH), and manganese (PDB entry code: 4X8D). The Tyr residue implicated in the catalytic cycle is indicated. Mn-binding residues are colored cyan.

performed on EgtB point mutants, a mechanism for C−S bond formation was proposed.358 In the presence of O2, the iron center of the substrate-bound complex forms an iron(III)superoxo species (Scheme 45). A single electron transfer from the thiol of γ-glutamyl cysteine and protonation of the resultant peroxide anion by an active site tyrosine affords the iron(III)5552

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48).378,379 Heterologous overexpression of the xia biosynthetic gene cluster in Streptomyces albus lead to the identification of the sulfadixiamycins.380 The structures of the sulfadixiamycins suggest that two equivalents of the pentacyclic xiamycin could capture sulfur dioxide, which may be formed in the course of sulfur primary metabolism, in a fashion similar to synthetic polymerization processes. Mutational analyses of the xia biosynthetic gene cluster revealed a candidate gene (xiaH) for sulfadixiamycin biosynthesis.380 A mutant strain lacking xiaH no longer produced the sulfa-dimers of xiamycin but was still able to produce the parent compound. Although the reaction could not be reconstituted in vitro, whole-cell biotransformation reactions with heterologously expressed xiaH were successful. Because XiaH also promotes the formation of N−C- and N−N-fused xiamycin dimers (dixiamycins) with the same substitution patterns,381 it was concluded that the formation of the C−S and N−S bonds proceeds through a radical mechanism. In fact, the fusion sites for dixiamycin and sulfadixiamycin biosynthesis (N-1, C-6, and C-21) can be rationalized by resonance stabilization of a xiamycin radical intermediate (Scheme 48). XiaH belongs to the large family of flavoenzymes, which mainly catalyze two-electron redox reactions. Although only rarely observed, some flavoenzymes are also capable of mediating single-electron transfers with formation of a flavin semiquinone radical. Perhaps the best-known examples are FAD-dependent monoamine oxidases, which oxidize amines to imines by one-electron oxidations that involve tyrosyl radicals.382,383 By analogy, sulfadixiamycin formation likely involves the formal abstraction of a hydrogen radical or a oneelectron oxidation to the radical cation, followed by deprotonation. The resulting resonance-stabilized radical intermediates may either pair to yield dixiamycins or react with sulfur dioxide. The sulfonyl radicals could then pair with another xiamycin radical.380

bifunctional enzyme catalyzes the methylation of histidine and subsequent sulfoxidation.361−363,365 Kinetic characterization showed that Egt1 from Neurospora crassa preferentially uses cysteine rather than the γ-glutamyl cysteine dipeptide as the sulfur donor for the sulfoxidation reaction.362 This result is consistent with a lack of an EgtC homologue in the genome of N. crassa. As with the mycobacterial EGT pathway, a C−S lyase (Egt2) removes the cysteine side chain to afford the mature compound.362,363 A similar moiety is found in ovothiol, a thiohistidine derivate initially isolated from sea urchin (Paracentrotus lividus) eggs366,367 and subsequently from various pathogenic protists such as Leishmania donovani and Trypanosoma cruzi.368,369 While the function of ovothiol is not yet understood, gene expression and metabolomic experiments in P. lividus link the molecule to the oxidative stress response during embryo development.370 Assays performed with cell-free extracts from the parasitic protist Crithidia fasciculata and radiolabeled ovothiol precursors implicated an oxygen-dependent enzyme in the conversion of cysteine and histidine to 5-histidylcysteine sulfoxide.371 In vitro reconstitution studies demonstrated that an EgtB/Egt1 homologue (OvoA) catalyzes the C-5 sulfoxidation of histidine using cysteine as the sulfur donor (Scheme 46).372 An evaluation of OvoA activity with different substrates suggested that the reaction mechanism parallels EgtB/Egt1-catalyzed C− S bond formation and indicated that the regioselectivity of OvoA (C-5 vs C-2 modification) is dictated by the structure of the sulfur acceptor.373,374 The product from the OvoA reaction is further processed to 5-thiohistidine and N-methylated at the imidazole ring to produce ovothiol, although the responsible enzymes have not been identified. 6.3. Flavoenzymes

In natural product biosynthesis, flavin-dependent oxygenases are known to promote a broad array of chemical redox transformations. The catalytic repertoire of the versatile flavin coenzymes includes both two- and one-electron transfers. Typically, dihydroflavins react with molecular oxygen to form flavin-4a-OOH adducts for various oxygenations. However, radical reactions promoted by flavoenzymes are also known.375 For the incorporation of sulfur, both avenues have been proposed. 6.3.1. Coelimycin. One or more flavoenzymes appear to be involved in the formation of the unusual 1,5-oxathiocane ring of coelimycin, an unusual yellow pigment produced by a Streptomyces coelicolor mutant.376 Bioinformatic analysis of the cpk gene locus and stable isotope-labeling experiments suggested that the coelimycin carbon backbone is assembled by a modular polyketide synthase. A plausible mechanism for the incorporation of N-actetyl cysteine would involve a bisepoxidated intermediate that could be attacked by the thiol group (Scheme 47). The epoxidation reactions could be catalyzed by the predicted gene products of cpkD, cpkH, and scF, which are all similar to known flavin-dependent epoxidases/dehydrogenases.376 6.3.2. Sulfadixiamycins. A radical-based C−S bond formation mediated by a flavoenzyme has been proposed for the biosynthesis of highly unusual diarylsulfone and sulfonamide antibiotics produced by a mangrove tree endophyte, Streptomyces sp. HKI0595. This endophytic streptomycete was previously shown to produce the indolosesquiterpene xiamycin (xia)377 by an unusual terpenoid cyclization sequence (Scheme

6.4. Tyrosinases

Tyrosinases are widely distributed type-3 copper-dependent enzymes that are mainly known to be involved in the biosynthesis of pigments.384−386 These dinuclear copper enzymes catalyze the four-electron oxidation of phenols to form ortho-quinones. In some cases these ortho-quinones can serve as electrophiles for the nucleophilic attack of thiols. It is notable that the active site of some tyrosinases contains a thioether linkage between a Cys and a copper-ligating His residue. This autoxidation reaction is proposed to conformationally restrain the His to improve copper binding.387 6.4.1. Pheomelanins. In humans and animals, tyrosinase catalyzes the hydroxylation of tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA) and its further oxidation into LDOPA quinone, a key intermediate in the biosynthesis of black/brown and red pigments named eumelanins and pheomelanins, respectively.384 The color of the pigments is strongly influenced by the incorporation of sulfur. In the presence of cysteine, the highly reactive DOPA quinone immediately reacts with the thiol to form cysteinyl-DOPA. After tyrosinase-mediated oxidation of the cysteinyl adduct into the corresponding quinone, intramolecular imine formation yields a benzothiazine heterocycle (Scheme 49). This building block and its regioisomers may polymerize to give the redcolored pheomelanins in human hair. Dimerization of the benzothiazine yields trichochromes, which are found not only in red human hair388 but also in skin, where they are in part 5553

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Scheme 45. Mechanism of Radical Sulfurization Catalyzed by EgtB

Scheme 46. Model for Ovothiol Biosynthesis

Scheme 47. Model for the Biosynthesis of Coelimycina

a

Following C−S bond formation, the opening of the second epoxide ring could proceed through a Payne rearrangement (a) or occur directly (b).

6.4.2. Grixazones. A tyrosinase homologue lacking ring hydroxylation activity enables the formation of a C−S bond in the biosynthesis of the Streptomyces griseus pigments grixazones A and B.391 These phenoxazinone derivatives are assembled from two 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) building blocks and N-acetylcysteine.392 Characterization of the grixazone (gri) biosynthetic pathway demonstrated that 3,4AHBAL is assembled from L-aspartate-4-semialdehyde and dihydroxyacetone phosphate by four proteins (Scheme 50).198,392 Two additional genes in the biosynthetic gene cluster, griF and griE, were predicted to be responsible for the

responsible for cellular photodamage as they function as endogenous UVA-photosensitizers.389 A similar heterocyclic system was found in cytotoxic red pigments (pheofungins) of an engineered mutant of Aspergillus nidulans impaired in posttranslational protein modification.390 Transcription analyses and gene knockout experiments indicated that pheofungins originate from phenolic compounds derived from orsellinic acid. Because tyrosinases are also widespread in the fungal domain, pheofungin biosynthesis likely involves the condensation of cysteine with ortho-quinones in analogy to the pheomelanin pathway.390 5554

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Scheme 48. Biosynthesis of Sulfadixiamycins A−C, Diarylsulfones, and Sulfonamides by Flavoenzyme-Mediated SO2 Capture

maturation of the 3,4-AHBAL-derived chromophore.393 The deduced gene products were homologous to tyrosinases (GriF) and tyrosinase-associated copper chaperones (GriE). Consistent with this prediction, heterologously produced GriE activated GriF by transferring copper ions, and a mutant lacking both genes accumulated 3,4-AHBAL. GriF was found to oxidize 3,4-AHBAL into the corresponding ortho-quinone imine, which condenses with a second equivalent of orthoquinone imine to form the phenoxazinone ring system. In the presence of N-acetylcysteine, grixazone A was formed by GriF in an in vitro assay. It was thus assumed that the Nacetylcysteine thiol undergoes a nonenzymatic conjugate addition to the ortho-quinone imine. The resulting thio conjugate of 3,4-AHBAL could be reoxidized by GriF and nonenzymatically coupled with another molecule of the orthoquinone imine to yield the mature grixazone (Scheme 50).393 6.4.3. Grape Reaction Product. Another prominent example of a tyrosinase involved in the formation of a C−S linkage is found in the production of the caftaric acid− glutathione conjugate, grape reaction product (GRP). During wine production, polyphenoloxidase oxidizes caftaric acid into

the corresponding ortho-quinone.394 In the presence of glutathione, the electrophilic ortho-quinone is converted to GRP (Scheme 51). Although GRP is the most abundant product, C-5 and C-6 glutathione conjugates and conjugates with other thiols have been observed.395 Consistent with this lack of specificity, in vitro experiments demonstrated that thiol addition is a nonenzymatic process.394,395 When the concentration of caftaric acid is greater than the concentration of the thiols in the grape juice, the ortho-quinone can readily react with various phenols (e.g., caftaric acid).396,397 The resultant dimers can be reoxidized by polyphenoloxidase and coupled to additional phenols, leading to the formation of brown pigments and loss of aroma compounds. This juice browning negatively impacts the quality of the wine and is commonly controlled by the addition of the polyphenoloxidase inhibitor SO2 or by the addition of a reductant (e.g., ascorbic acid) that can reduce the ortho-quinones back to caftaric acid. 5555

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Scheme 49. Model for the Biosynthesis of Eumelanins and Pheomelaninsa

a Tyrosinases generate highly reactive ortho-quinones that are readily attacked by cysteine. The fungal pigment pheofungin B may be formed by an analogous process.

Scheme 50. Model for the Biosynthesis of Grixazone

7.1. Thioether-Forming rSAMs

7. RADICAL S-ADENOSYLMETHIONINE ENZYMES Radical S-adenosylmethionine (rSAM) proteins form a highly diverse family of enzymes catalyzing a wide range of radical reactions.398 Members catalyze the reductive cleavage of SAM to methionine and the highly reactive 5′-deoxyadenosyl radical using a [4Fe-4S] cluster (Scheme 52). This radical is then used to perform the wide array of difficult transformationsoften the functionalization of unactivated C−H bondsthat are characteristic of the family. Although rSAM enzymes are involved in the biosynthesis of multiple classes of natural products, C−S bond-forming rSAM enzymes are scarce.

A subset of rSAM enzymes catalyze C−S bond formation. Characterized sulfur-inserting rSAMs are responsible for the formation of the thioether linkages of important cofactors (biotin and lipoic acid) and for the methylthiolation of tRNA and ribosomal proteins.399 A remarkable feature of such enzymes is their use of a second iron−sulfur cluster in C−S bond formation. This auxiliary cluster is thought to serve as a sulfur source for the cofactor synthesizing enzymes, whereas it is thought to bind the methylthio moiety prior to reaction with the substrate radical in methylthiolating enzymes. Recent studies have implicated rSAM proteins in C−S bond formation in two classes of natural products, namely, albomycin and the sactipeptides. In both instances the sulfur-inserting rSAM is thought to use an auxiliary iron−sulfur cluster to catalyze C−S bond formation. 7.1.1. Albomycin. Albomycin is a Trojan horse antibiotic from Streptomyces spp. that employs siderophore transport systems to enter bacterial cells.400−402 The molecule is composed of a potent iron chelator ferrichrome linked to a derivative of SB-217452, an aminoacyl-tRNA synthetase inhibitor (Scheme 53). After entering bacterial cells, the conjugate is hydrolyzed by cellular peptidases, thus releasing the bioactive nucleoside antibiotic.403 Albomycin features a tetrahydrothiophene ring that is essential for its antibiotic potency; it is remarkable that substitution of the sulfur atom for oxygen eliminates activity.404 The albomycin S-heterocycle is reminiscent of the tetrahydrothiophene ring of biotin, which is installed by the rSAM enzyme BioB. The stepwise C−S bond formation catalyzed by BioB has been studied in detail.398 In brief, BioB cleaves SAM units to generate 5′-deoxyadenosyl radicals, which abstract hydrogens from the methyl and methylene substituents of a cyclic urea intermediate, and the [2Fe-2S]-cluster bound to BioB serves as the sulfur donor (Scheme 53). It is plausible that 5556

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Scheme 51. Production of Grape Reaction Product in Winemaking; G-SH, Glutathione; PPO, Polyphenoloxidase

domain.235,236,417−419 Sactionine synthetase mutants lacking these auxiliary 4Fe-4S clusters are not able to catalyze thioether formation despite being able to perform SAM cleavage. Two roles have been proposed for the role of the auxiliary 4Fe-4S cluster(s) in thioether formation (Scheme 54). In the initial reconstitution report of the subtilosin biosynthetic enzyme, AlbA, it was demonstrated that the UV−vis absorption spectrum of the auxiliary 4Fe-4S cluster changes when the precursor peptide is present.417 This was used as evidence to propose that the cysteine thiol of the thioether linkage is ligated to the auxiliary metal center, which acts as an electron sink for the formation of the thioether between the carbon-centered radical and the cysteine thiol (Scheme 54).417−419 However, the direct ligation mechanism does not provide an explanation for the presence of both D- and L-sactionine linkages in subtilosin A and thuricin CD.410,420 The second mechanism proposes that the auxiliary 4Fe-4S cluster is involved in the oxidation of the carbon-centered radical to an N-acyliminium ion (Scheme 54).235,413 This planar intermediate is then attacked at either the si- or re-face to generate either the L- or D-sactionine linkage, respectively. The face of attack is proposed to be controlled by the flexibility of the substrate, akin to the mechanism observed in lanthionine formation (see section 4.1).233 While the current data does not rule out either mechanism, it is of note that the two auxiliary 4Fe-4S clusters in the SPASM-containing rSAM protein anaerobic sulfatasematurating enzyme (anSME) are thought to be involved in substrate oxidation.421 An identical thioether linkage is found in the cyclothiazomycin group of thiopeptides (Scheme 55).422−424 This linkage is formed between a cysteine thiol and a serine residue through an unknown mechanism. As the biosynthetic gene cluster lacks any genes with homology to rSAM enzymes, it is evident that this moiety is not installed in an analogous fashion to the sactionine linkages of the sactipeptides.424,425 The current biosynthetic proposal involves the formation of a dehydroalanine by a lanthipeptide dehydratase (see section 4.1) encoded in the biosynthetic gene cluster, followed by a Michael addition and subsequent rearrangement (Scheme 55).425 Notably, this putative Michael addition is not catalyzed by a lanthipeptide cyclase as one is not present in the biosynthetic cluster. 7.1.3. γ-Subunit of Quinohemoprotein. Outside of RiPP biosynthesis, a SPASM domain-containing rSAM enzyme is also involved in C−S bond formation in the posttranslational maturation of quinohemoprotein amine dehydrogenase (QHNDH).426−428 QHNDH is composed of three proteins, QhpA−C, that together catalyze the oxidative deamination of

sulfur could be introduced into the carbon backbone of the albomycin precursor molecule by an analogous mechanism.48 Indeed, analysis of the albomycin (abm) biosynthetic gene cluster showed two candidate genes, abmM and abmJ, coding for rSAM enzymes.405 The predicted gene product of abmM is similar to BioB and shows binding motifs for [4Fe-4S] and [2Fe-2S] clusters. Sulfur could be delivered by the putative cysteine desulfurase AbmD, which appears to represent an important link between primary and secondary sulfur metabolic pathways; generally, cysteine desulfurases are involved in the assembly of Fe−S clusters in bacteria.406 Interestingly, pathways responsible for increasing homocysteine levels were shown to positively impact albomycin production.407 Yet, the exact mechanism of sulfur incorporation remains to be elucidated. 7.1.2. Sactipeptides. The sactipeptides (sulfur-to-α carbon-linked peptides) are a subclass of RiPP natural products bearing thioether linkages between a cysteine thiol and the unactivated α-carbon of an acceptor amino acid (Scheme 54).22 In analogy to lanthionine rings, this moiety has recently been named a sactionine linkage.234 To date, six sactipeptides have been reported: subtilosin A, subtilosin A1, sporulation killing factor (SKF), thuricin CD (composed of two peptides, thuricin α and thuricin β), and thurincin H.206,299,408−412 A seventh sactipeptide, annotated as a member of the uncharacterized “six cysteines in forty-five” (SCIFF) group, was recently partially biosynthesized in vitro with recombinant enzymes.413,414 Initial studies regarding the biosynthesis of sactipeptides demonstrated that a member of the rSAM protein family was required for production.415,416 As many characterized rSAM proteins catalyze C−H bond abstraction,398 it was proposed that the rSAM enzyme found in all sactipeptide BGCs is responsible for sactionine formation.410,415 In the last five years, the activities of multiple sactionine synthases have been reconstituted in vitro.413,417−419 As predicted bioinformatically, all of the enzymes bind a 4Fe-4S cluster with a canonical triple cysteine motif (CxxxCxxC) that is a hallmark of rSAM enzymes. In all of the sactionine synthases, this rSAM 4Fe-4S cluster is used to reductively cleave SAM to form methionine and a 5′-deoxyadenosyl radical (5′-dAdo·; Scheme 54). Using peptide substrates that were deuterium-labeled at the α-carbon of the acceptor amino acid, it was demonstrated that this 5′dAdo extracts the Cα hydrogen to initiate thioether bond formation.235,236 In addition to the rSAM 4Fe-4S cluster, reconstituted sactionine synthases contain up to two additional 4Fe-4S clusters that are bound by conserved cysteine residues in their C-terminal SPASM (subtilosin, thuricin H, and SCIFF synthase) or Twitch (SKF and thuricin CD synthase) 5557

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Scheme 52. Model of Reductive SAM Cleavage by rSAM Enzymes

Scheme 53. Model for the Biosynthesis of the Trojan Horse Antibiotic Albomycin, Likely Involving Putative rSAM Enzymes AbmM and/or AbmJ; Box: Biotin Biosynthesis Involving an rSAM Enzyme with a [2Fe-2S] Cluster

8. NONENZYMATIC C−S BOND FORMATIONS

aliphatic amines. This protein complex is found in a variety of Gram-negative and Gram-positive bacteria. The γ-subunit of QHNDH, QhpC, is a small protein (∼9 kDa) bearing three thioether cross-links at the β-carbon and γ-carbon of aspartate and glutamate residues, respectively (Scheme 56).429,430 These linkages are required to provide structure to the small peptide. A recent report demonstrated that a homologue of the sactionine synthase, QhpD, is responsible for installing these linkages.428 In addition to the aforementioned thioether bridges, QhpC also contains a fourth thioether linkage formed between a cysteine thiol and the indole of an oxidized tryptophan residue, known as a cysteine tryptophylquinone (CTQ) moiety.429,430 The biosynthetic strategy for the formation of the C−S bond in CTQ is unknown.427

As outlined in the preceding sections, several enzymes involved in the biosynthesis of organosulfur natural products produce highly reactive intermediates that have the potential to scavenge sulfur-bearing molecules. Indeed, in some instances it is unclear whether the C−S bond in question is formed enzymatically or due to the innate reactivity of the biosynthetic intermediate. For example, the inability to identify a S-transferase in brassinin biosynthetic pathways suggests that glutathione conjugation may result from a spontaneous reaction with the electrophilic isothiocyanate moiety (see section 3.1.6). In this section, a selection of nonenzymatic additions to secondary metabolites will be presented. Apart from nucleophilic additions, C−S 5558

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Scheme 54. Model for rSAM-Catalyzed Thioether Formation in Sactipeptide Biosynthesisa

a

The two proposals for the role of the auxiliary 4Fe-4S clusters in C−S bond formation are displayed.

Scheme 55. Model for Thioether Formation in Cyclothiamycin Biosynthesis

bond-forming radical reactions are conceivable, which may be initiated by redox cycling or photoactivation by UV light.

mainly composed of sugar units, allows the binding of calicheamicin to DNA and intercalation of the enediyne “warhead” between base stacks. The trisulfide bond of the “prodrug” of the highly cytotoxic natural product is cleaved by reduction or attack of a nucleophile, which initiates an intramolecular conjugate addition of the remaining sulfide to the enone system (Scheme 57). The transition of sp2 to sp3 hybridization at the quaternary, sulfur-substituted carbon

8.1. Nonenzymatic Conjugate Addition

8.1.1. Enediyne Warhead Activation. Perhaps one of the best-known uncatalyzed C−S bond formations takes place in the context of the trigger reaction leading to calicheamicinmediated DNA double-bond scission.431,432 The “homing” unit, 5559

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Scheme 56. Structure of Mature QhpC

Scheme 57. Nonenzymatic Sulfide Conjugate Addition That Sets the Stage for Bergman or Myers−Saito Cyclizations of Calicheamicin and Neocarzinostatin, Respectivelya

a

The resulting diradical intermediate induces double-strand (ds) DNA scissions.

derive from methionine and that the methanethiol residue was incorporated intact. A mechanistically plausible route was proposed that would involve the Michael addition of the thiol to urdamycin A, followed by rearomatization and reoxidation to the quinone with molecular oxygen (Scheme 58).434 Analyses of the urdamycin435 and BE-7585A299 biosynthetic gene clusters did not provide any clues about candidate genes potentially required for C−S bond formation. Thus, it was also taken into consideration that the conjugate addition might not require any biocatalyst. To test this hypothesis, urdamycin A was incubated with either 2-thio-D-glucose or glucose in aqueous buffer at pH 8.0.299 HPLC-MS monitoring of the reaction revealed that only in the presence of the thiosugar a new compound was formed, which appeared to be the expected thio adduct. By analogy to the model reaction with urdamycin A and 2-thio-D-glucose, the C−S bond of BE-7585A would result from the conjugate addition of the preformed thiodisaccharide on the angucycline core (Scheme 58). It is remarkable that no other thio derivatives of urdamycin and BE-7585A have been detected in the broths of the producing organisms, not even the thioether that would result from the nucleophilic attack of a thiomonosaccharide formed during BE-7585A biosynthesis. Although is it possible that such a monosaccharide adduct could be transformed into BE-7585A after the Michael addition, the absence of any other thio

increases the strain on the enediyne ring and decreases the distance between the alkyne moieties. The highly strained intermediate readily undergoes a Bergman cyclization, thereby alleviating the torsional strain. The resulting aryl diradical abstracts hydrogen from the DNA backbone, thus inducing double-strand scissions (Scheme 57). An analogous transformation occurs in the activation of the enediyne natural product neocarzinostatin, albeit through a different mechanism.431,432 The 1,8 conjugate addition of a cellular thiol yields the highly reactive cummulene intermediate, which undergoes a Myers−Saito cyclization (Scheme 57). As with calicheamicin, the diradical product of this cyclization causes double-strand DNA breaks. Notably, neocarzinostatin can also be activated by a base-catalyzed intramolecular addition in the absence of a thiol nucleophile. 8.1.2. Urdamycin E and BE-7585A. Urdamycin E from Streptomyces f radiae433 and BE-7585A from Amycolatopsis orientalis (see section 5.3.2) are angucyclic polyketide antibiotics with unusual sulfur modifications. In both cases, C−S bonds between a benz[a]anthraquinone core and a thiol are installed. Feeding experiments with methyl-13C-labeled methionine, selenomethionine, and selenoethionine led to the formation of methyl-13C-labeled urdamycin E, seleno-urdamycin E, and its ethyl homologue, respectively. Thus, it was concluded that the sulfur and the methyl group of urdamycin E 5560

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Scheme 58. Tentatively Nonenzymatic Conjugate Additions of Thio Nucleophiles to Angucyclines Involved in the Formation of the Antibiotics Urdamycin A and BE-7585A

culture.437 The finding that the ralfuranone scaffold readily reacts with sulfur nucleophiles including glutathione may have implications for plant infections involving reactive oxygen species.

adducts suggests that the reaction is controlled in some way. It is conceivable that either appropriate S-nucleophiles are shuttled to the reactive angucycline core or that enzyme complexes of polyketide synthase components and sugartailoring enzymes prevent the attack of alternative sulfur compounds. 8.1.3. Ralfuranone D. Yet another type of Michael acceptor sets the stage for nonenzymatic thiol conjugate additions to yield thioethers in ralfuranones, a family of arylsubstituted furanones isolated from the plant pathogenic bacterium Ralstonia solanacearum (Scheme 59).436,437 Specifically, thiol and methyl thioether derivatives (e.g., ralfuranone D) of the previously characterized ralfuranone B436 were detected. Through analysis of the ralfuranone biosynthetic gene cluster,438 feeding experiments, and in vitro studies it was concluded that the C−S bond formation does not involve enzyme catalysis. Stable-isotope-labeling experiments with L[methyl-13C2H3] methionine indicated that the methyl thio group was introduced intact into ralfuranone D. Addition of various thiols to R. solanacearum cultures yielded the corresponding thio conjugates, including the cysteinyl and glutathione adducts. Furthermore, a new ralfuranone thioether derivative, an analogue of ralfuranone D, was obtained by adding ethanethiol to the crude extract of a R. solanacearum

8.2. Nonenzymatic Addition

8.2.1. Cyslabdan. Cyslabdan is a diterpene natural product bearing a N-acetylcysteine residue isolated from Streptomyces cyslabdanicus K04-0144.439−441 While cyslabdan alone does not display significant antibacterial activity, it potentiates the activity of carbapenem antibiotics against methicillin-resistant Staphylococcus aureus by >1000-fold.440−442 Genome mining for the biosynthetic gene cluster identified four genes cldA−D, which when expressed in a heterologous host lead to the production of cyslabdan A.443 Additionally, a new compound bearing an epoxide was detected. As cldC encoded a protein with homology to cytochrome P450 enzymes, a version of the cld biosynthetic gene cluster lacking this gene was created and screened for production of cyslabdan derivatives. Consistent with the functional annotation of CldC, the reduced diterpene derivative was obtained (Scheme 60). The discovery that the biosynthesis of cyslabdan A proceeded through an epoxide intermediate suggested that the C−S bond might be formed nonenzymatically likely through the addition of mycothiol to 5561

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the highly reactive epoxide. Indeed, a deletion strain lacking the mycothiol amide hydrolase mca produced the mycothiolconjugated diterpene rather than cyslabdan A. Although an enzymatic route to C−S bond formation has not been ruled out for cyslabdan biosynthesis, the available data strongly suggest that mycothiol conjugation will occur spontaneously. The spontaneous nucleophilic attack of an epoxide is likely involved in the biosynthesis of the spithioneines. These ergothioneine-conjugated pyrrolizidine alkaloid natural products were isolated from the marine actinomycete Streptomyces spinoverrucosus strain SNB-048.444 Given their structure, it was proposed that the spithioneines arose from the conjugation of ergothioneine to the epoxide-containing precursor bohemamine (Scheme 61). Indeed, when bohemamine and ergothioneine were coincubated under basic conditions, spithioneine A was formed. As the biosynthetic gene cluster for these natural products has not been identified, it is unclear whether the conjugation reaction will proceed nonenzymatically in vivo. 8.2.2. Paulomycin S-Conjugates. Paulomycins are a group of isothiocyanate-containing antibiotics originally isolated from Streptomyces paulus.445−447 Large-scale fermentations of S. paulus facilitated the isolation and structural characterization of a series of paulomycin analogues (Scheme 62) where the isothiocyanate is conjugated to N-acetylcysteine (paldimycins and antibiotic 273a2) or hydrogen sulfide (U-77,802 and U-77,803).448−450 Recently, the paulomycin biosynthetic gene cluster was identified and the biosynthetic pathway was partially characterized.451−453 Although many aspects of biosynthesis remain unclear, the lack of an obvious candidate enzyme for isothiocyanate conjugation suggests that these congeners are spontaneously generated. In support of this proposal, the paldimycins are readily synthesized from the corresponding paulomycin in vitro with the addition of N-acetylcysteine and a mild base.448

Scheme 59. Vinylogous Thiol Addition Leads to Ralfuranone D Formation

Scheme 60. Cyslabdan Biosynthesis Proceeds through an Epoxide Intermediate; MSH; Mycothiol

Scheme 61. Possible Origin of Spithioneines

8.3. Photochemical and Radical-Mediated Thioconjugation

8.3.1. Panphenazines. Radical-based thioconjugation reactions have been observed for the phenazine chromophore, which is widespread among bacteria.454 Genome mining and metabolic profiling of the rare actinomycete Kitasatospora sp. led to the discovery of prenylated phenazines along with pantetheine-S-conjugates of phenazine-1-carboxylic acid (PCA).455,456 In vitro experiments showed that these so-called panphenazines could be formed in two ways, chemically induced and photoinduced, both involving phenazine radicals (Scheme 63). Phenazine radicals are formed during redox cycling processes in vivo, which involve single-electron transfers.454 PCA reacts with diverse biogenic thiols under radical-forming conditions, which provides a plausible model for irreversible glutathione depletion in cells in the presence of the bacterial phenazine pyocyanine.457 Furthermore, the phenazine UV absorption maxima at 250 and 370 nm enable photoexcitation by UV light. Indeed, irradiation of PCA in the presence of pantetheine led to the formation of panphenazines.456 Hence, it is conceivable that photoactivation of suitable (hetero)aromatic natural products may lead to thioconjugates.

responsible biosynthetic pathways and enzymes. Despite the diversity of the biosynthetic enzymes involved and of the linkages installed, common themes have emerged. For example, the biosynthetic logic for C−S bond formation first identified in the modification of tRNA and cofactor biosynthesis has been uncovered in the biosynthesis of secondary metabolites. Furthermore, pathways for the detoxification of reactive electrophilic compounds have been coopted by secondary metabolite biosynthetic enzymes to introduce sulfur from cellular thiols (e.g., glutathione and mycothiol). Similar approaches to C−S bond formation can be found in

9. CONCLUSIONS AND OUTLOOK Nature has evolved varied strategies to install carbon−sulfur bonds in natural products, and, over the last few decades, substantial progress has been made toward elucidating the 5562

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Scheme 62. Structures of Paulomycin Thiol Conjugates; NAC; N-acetylcysteine

Scheme 63. Structures of Naturally Occurring S-Conjugates of Phenazine-1-carboxylic Acid (PCA); Model for Radical-Based Thioconjugation of Phenazines

alliin, the trisulfide trigger of calicheamicin); however, there are many natural products that were omitted as their biosynthesis is completely enigmatic. For example, in addition to 6thioguanine and thioviridamide, there are a handful of thioamide-containing natural products for which the corresponding biosynthetic enzymes are unknown (Scheme 64).460−463 The current understanding of the biocatalysts involved in the production of organosulfur compounds may guide the investigation of these obscure pathways and provide a

taxonomically distant organisms, potentially arising independently or from horizontal gene transfer, as is the case for isopenicillin N synthase genes.458,459 Although the body of knowledge on the biosynthesis of sulfur-containing natural products has grown rapidly in recent years, many questions remain regarding the enzymes, precise mechanisms, and biosynthetic building blocks involved. Where possible, we have attempted to highlight these knowledge gaps (e.g., thiosugar biosynthesis, the origin of the allyl moiety of 5563

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Scheme 64. Thioamide-Containing Natural Products Where the Biosynthesis of the C−S Bond Remains Enigmatic

foundation for the discovery of novel sulfur-bearing natural products.464 Moreover, the many mechanisms involved in C−S bond formation may inspire synthetic chemists in the synthesis of complex thio compounds.

mining and elucidation of natural product biosynthetic pathways from phytopathogenic bacteria. Christian Hertweck is the Head of Department at the Leibniz Institute for Natural Product Research and Infection Biology (HKI), a Full Professor at the Friedrich Schiller University Jena, and an elected member of the National Academy of Sciences (Leopoldina). He obtained his Ph.D. in Bioorganic Chemistry in 1999 (supervisor Wilhelm Boland) at the University of Bonn and the Max Planck Institute for Chemical Ecology before his postdoctoral work at the University of Washington, Seattle, as a Humboldt fellow of Heinz G. Floss and Bradley S. Moore. His research focuses on the biosynthesis of microbial natural products and their roles in interspecies interactions. In 2015 he was awarded the Leibniz Prize in recognition of his contribution to the field.

AUTHOR INFORMATION Corresponding Author

*Tel.: +49 3641 532 1100. Fax: +49 3641 532 0804. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Kyle Dunbar obtained his B.Sc. in Chemical Biology from the University of California, Berkeley. In 2014, he received his Ph.D. in Chemistry from University of Illinois at Urbana−Champaign under the supervision of Douglas Mitchell. His Ph.D. work focused on the biosynthesis of azoline heterocycles in ribosomal natural products. Following a short postdoctoral position at the University of Illinois at Urbana−Champaign, Kyle joined the group of Christian Hertweck at the Leibniz Institute for Natural Product Research and Infection Biology as an Alexander von Humboldt postdoctoral fellow. His current research focuses on the molecular characterization of interspecies interactions.

ACKNOWLEDGMENTS We thank Florian Kloss for valuable discussions during the initial planning phase and Evelyn Molloy for the critical review of the manuscript. Financial support by the Alexander von Humboldt Foundation (for K.L.D.) and the National Academy of Sciences (Leopoldina) (for D.H.S.) is gratefully acknowledged. REFERENCES (1) Wächtershäuser, G. From Volcanic Origins of Chemoautotrophic Life to Bacteria, Archaea and Eukarya. Philos. Trans. R. Soc., B 2006, 361, 1787−1806. (2) Richter, M. Functional Diversity of Organic Molecule Enzyme Cofactors. Nat. Prod. Rep. 2013, 30, 1324−1345. (3) Cipollone, R.; Ascenzi, P.; Tomao, P.; Imperi, F.; Visca, P. Enzymatic Detoxification of Cyanide: Clues from Pseudomonas Aeruginosa Rhodanese. J. Mol. Microbiol. Biotechnol. 2008, 15, 199− 211. (4) Fahey, R. C. Glutathione Analogs in Prokaryotes. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 3182−3198. (5) Shigi, N. Biosynthesis and Functions of Sulfur Modifications in tRNA. Front. Genet. 2014, 5, 67. (6) Begley, T. P.; Xi, J.; Kinsland, C.; Taylor, S.; McLafferty, F. The Enzymology of Sulfur Activation During Thiamin and Biotin Biosynthesis. Curr. Opin. Chem. Biol. 1999, 3, 623−629. (7) Mueller, E. G. Trafficking in Persulfides: Delivering Sulfur in Biosynthetic Pathways. Nat. Chem. Biol. 2006, 2, 185−194.

Daniel H. Scharf received his Diploma degree (M.S. equiv) in Biology from the Friedrich Schiller University Jena, Germany, in 2008. He then continued his Ph.D. studies at the Leibniz Institute for Natural Product Research and Infection Biology under the supervision of Axel A. Brakhage. His Ph.D. researched focused on the gliotoxin biosynthetic pathway in Aspergillus f umigatus. After a postdoc in his thesis laboratory, he joined the group of Georgios Skiniotis at the Life Sciences Institute, University of Michigan, in 2016. His current research interests concern the structural and mechanistic characterization of polyketide synthases and nonribosomal peptide synthetases. Agnieszka Litomska was born in Lodz, Poland, and received her M.Sc. degree in Biotechnology, with specialization in Molecular Biotechnology and Technical Biochemistry, from Lodz University of Technology in 2012. She is currently a Ph.D. student in the laboratory of Christian Hertweck at the Leibniz Institute for Natural Product Research and Infection Biology in Jena, Germany. Her research focuses on genome 5564

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