Biosynthesis of Lincosamide Antibiotics: Reactions Associated with

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

pubs.acs.org/accounts

Biosynthesis of Lincosamide Antibiotics: Reactions Associated with Degradation and Detoxification Pathways Play a Constructive Role Daozhong Zhang,†,§ Zhijun Tang,†,§ and Wen Liu*,†,‡ †

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence on Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China ‡ Huzhou Center of Bio-Synthetic Innovation, 1366 Hongfeng Road, Huzhou 313000, China CONSPECTUS: Natural products typically are small molecules produced by living organisms. These products possess a wide variety of biological activities and thus have historically played a critical role in medicinal chemistry and chemical biology either as chemotherapeutic agents or as useful tools. Natural products are not synthesized for use by human beings; rather, living organisms produce them in response to various biochemical processes and environmental concerns, both internal and external. These processes/concerns are often dynamic and thus motivate the diversification, optimization, and selection of small molecules in line with changes in biological function. Consequently, the interactions between living organisms and their environments serve as an engine that drives coevolution of natural products and their biological functions and ultimately programs the constant theme of smallmolecule development in nature based on biosynthesis generality and specificity. Following this theme, we herein review the biosynthesis of lincosamide antibiotics and dissect the process through which nature creates an unusual eight-carbon aminosugar (lincosamide) and then functionalizes this common high-carbon chain-containing sugar core with diverse L-proline derivatives and sulfur appendages to form individual members, including the clinically useful anti-infective agent lincomycin A and its naturally occurring analogues celesticetin and Bu-2545. The biosynthesis of lincosamide antibiotics is unique in that it results from an intersection of anabolic and catabolic chemistry. Many reactions that are usually involved in degradation and detoxification play a constructive role in biosynthetic processes. Formation of the trans-4-propyl-L-proline residue in lincomycin A biosynthesis requires an oxidation-associated degradation-like pathway composed of heme peroxidase-catalyzed ortho-hydroxylation and non-heme 2,3-dioxygenase-catalyzed extradiol cleavage for L-tyrosine processing prior to the building-up process. Mycothiol (MSH) and ergothioneine (EGT), two small-molecule thiols that are known for their redox-relevant roles in protection against various endogenous and exogenous stresses, function through two unusual S-glycosylations to mediate an eight-carbon aminosugar transfer, activation, and modification during the molecular assembly and tailoring processes in lincosamide antibiotic biosynthesis. Related intermediates include an MSH Sconjugate, mercapturic acid, and a thiomethyl product, which are reminiscent of intermediates found in thiol-mediated detoxification metabolism. In these biosynthetic pathways, “old” protein folds can result in “new” enzymatic activity, such as the DinB-2 fold protein for thiol exchange between EGT and MSH, the γ-glutamyltranspeptidase homologue for C−C bond cleavage, and the pyridoxal-5′-phosphate-dependent enzyme for diverse S-functionalization, generating interest in how nature develops remarkably diverse biochemical functions using a limited range of protein scaffolds. These findings highlight what we can learn from natural product biosynthesis, the recognition of its generality and specificity, and the natural theme of the development of bioactive small molecules, which enables the diversification process to advance and expand small-molecule functions.

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INTRODUCTION

such as signal transduction, competition, and adaptation. On the other hand, cells can utilize (neutralize) natural or unnatural (toxic) substances and degrade them into alternative substrates and energy sources for biomass improvement and natural product biosynthesis.3,4 Beyond these substance- and energy-related exchanges, the intersection of anabolic and catabolic chemistry is extremely interesting. In particular, reactions usually associated with degradation and detoxification

Living organisms possess a remarkable capacity to interface with their internal and external environments in a variety of ways. Benefiting from a marvelous diversity in catalysis, simple substrates, including short carboxylates, amino acids, and sugars, can be converted to various building blocks. Enzymatic permutation/combination and polymerization of these building blocks are often followed by postsynthetic modifications, leading to the production of numerous bioactive small molecules1,2 that can modulate or participate in various biochemical processes and combat environmental stresses © XXXX American Chemical Society

Received: March 24, 2018

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DOI: 10.1021/acs.accounts.8b00135 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. Chemical structures of (A) lincosamide antibiotics and (B) representative ALDP-containing natural products, including PBDs and hormaomycins. Related building blocks are highlighted in color, including the C8 aminosugar unit lincosamide (orange), ALDP residues (green), and sulfur appendages (blue).

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OVERVIEW OF BIOSYNTHETIC ORIGIN Insights into the biosynthetic origin of lincosamide antibiotics were initially gained from cultures of the lincomycin Aproducing S. lincolnensis strain with various isotope-labeled precursors, which showed that the shared lincosamide core is likely formed through condensation of a three-carbon (C3) unit and a five-carbon (C5) unit, both of which can be derived from 11 D-glucose. In contrast with celesticetin and Bu-2545, which share a proteinogenic L-proline residue, the PPL residue of lincomycin A is non-proteinogenic and arises primarily from the rearrangement of L-tyrosine.12 Similar results were found in labeling studies of the cyclodepsipeptides hormaomycins (microbial hormones for antibiotic production and aerial mycelium formation in actinomycetes)13 and the pyrrolobenzodiazepines (PBDs) (sequence-specific DNA alkylating agents with remarkable anticancer activity), such as anthramycin, porothramycin, sibiromycin, and tomaymycin (Figure 1B).14,15 These natural products are distinct from lincomycin A in both structure and biological activity but share a PPL-like L-tyrosinederived 4-alkyl-L-(dehydro)proline (ALDP) residue that varies in the oxidation state, length, and extent of functionalization of the carbon side chain. S-Adenosyl-L-methionine (SAM) is likely the methyl donor for both N- and S-methylations during lincomycin A biosynthesis.16 The origin of the sulfur atom within the methylmercapto group has been the subject of much speculation but remained unclear until recently. Following the steps for cloning of resistance genes,17 the entire biosynthetic gene cluster of lincomycin A (lmb) was identified and sequenced in the S. lincolnensis strains 78−11 and ATCC 25466.18,19 Related lmb genes were applied as probes to identify the celesticetin biosynthetic gene cluster (ccb),20 permitting a comparative analysis of different lincosamide antibiotics in terms of the generality (e.g., for lincosamide formation and associated molecular assembly logic) and specificity (e.g., for aminoacyl preparation and sulfur

processes can be utilized and programmed to create extraordinary biosynthetic pathways for functional small molecules, as exemplified here by lincosamide antibiotics (Figure 1A),5 which share lincosamide, an eight-carbon (C8) aminosugar but differ in the aminoacylation and sulfur functionalization of this unusual high-carbon chain-containing sugar unit. Lincosamide antibiotics include the therapeutic antibacterial drug lincomycin A and its naturally occurring analogues celesticetin and Bu-2545 (Figure 1A). Lincomycin A, which is characterized by an N-methylated trans-4-propyl-L-proline (PPL, 15) residue conjugated to lincosamide via an amide linkage and a methylmercapto group appended to the sugar moiety at C1, was isolated from Streptomyces lincolnensis, a soil Gram-positive actinobacterium native to Lincoln, Nebraska.6 Since its application in the treatment of children with acute pharyngitis or tonsillitis arising from hemolytic streptococcal infection in 1963,7 this antibiotic has been widely used as an antimicrobial drug for over half a century. Although rarely prescribed today because of adverse effects and toxicity, it is reserved for patients allergic to penicillin or for treating bacteria that have developed resistance. Celesticetin, which was isolated from Streptomyces caelestis, has a lincosamide core central to a 4unsubstituted N-methyl-L-proline residue and a salicyclic moiety through an S-linked two-carbon alcohol unit.8 Interestingly, the Streptomyces strain H230-5 produces Bu2545,9 which is a structural hybrid between lincomycin A and celesticetin and results from permutation of the two decorations of the octose unit. Consequently, the structural similarity of these lincosamide antibiotics to the 3′ end of LPro-Met-tRNA or deacyl-tRNA allows them to target the peptidyltransferase center of the 50S large subunit of the bacterial ribosome, thereby perturbing protein synthesis at the initial stage of the elongation cycle.10 B


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Figure 2. Comparative analysis of the biosynthetic gene clusters of lincomycin A and celesticetin. The functions of genes, either common or specific, are annotated by rectangular blocks with different colors at the bottom. Sequence identity (ID) is indicated between each pair of homologous genes.

Figure 3. Biosynthetic pathway of the C8 aminosugar unit. The carbon atoms of the C3 and C5 units are colored in both the precursors and the product. The proposed mechanism of LmbR-catalyzed transaldol reaction is shown in the dashed rectangle.

decoration) of their biogenesis (Figure 2). Significant progress has been made over the past decade in accessing the process by

which nature creates these remarkable molecules. By assaying related enzymatic activities in vitro, an unusual paradigm for C8 C


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Figure 4. Formation of the PPL moiety of lincomycin A. The oxygen atoms from H2O2 (blue) and O2 (red) are labeled. (A) Biosynthetic pathways of PPL. The shared reactions in the formation of ALDP residues are shaded. (B) Heme peroxidase-catalyzed ortho-hydroxylation. (C) Non-heme dioxygenase-catalyzed oxidative extradiol cleavage.

with R5P for C3 unit transfer through aldol condensation (Figure 3). Several genes shared by the lmb and ccb clusters are homologous with those involved in nucleoside diphosphate (NDP)-deoxysugar biosynthesis, suggesting that the formation of a lincosamide core involves sugar intermediates in an NDPactivated form.22 Further bioinformatics-based analysis revealed that additional gene products share sequence homology with those involved in the pathway for NDP-heptose formation, which requires the successive processing of a heptulose 7phosphate precursor by 1,2-isomerization to form heptose 7phosphate, anomeric phosphorylation to give heptose 1,7biphosphate, and 7-dephosphorylation to form heptose 1phosphate before nucleotidyl transfer.23 Consistently, the formation of an NDP-octose was confirmed to share a similar pathway involving isomerase, kinase, and phosphatase activities (Figure 3).22 In the presence of LmbN, a bifunctional protein possessing a C-terminal domain homologous to S7P isomerase, intermediate 4 undergoes C1−C2 isomerization to produce Derythro-D-gluco-octose 8-phosphate (5), which can be anomerically phosphorylated by the kinase LmbP to yield octose 1,8biphosphate (6). LmbK is a phosphatase and catalyzes the 8dephosphorylation of 6 to form octose 1-phosphate (7), which is then subjected to nucleotidyl transfer catalyzed by LmbO, a nucleotidylyltransferase that functions specifically in the

sugar formation was uncovered; in particular, proteins homologous to those typically involved in degradation and detoxification pathways were observed, paving the way to an appreciation of their constructive role in the diversification and functionalization of the common lincosamide core.

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FORMATION OF A SHARED LINCOSAMIDE CORE

Both the lmb and ccb clusters contain a gene coding for a putative transaldolase (e.g., LmbR in lincomycin A biosynthesis), supporting the conclusion resulting from isotopelabeling experiments that the C8 skeleton of lincosamide is formed through condensation of a pentose 5-phosphate with a C3 unit in a transaldol reaction.11 As expected, LmbR can use either D-fructose 6-phosphate (F6P, 2) or D-sedoheptulose 7phosphate (S7P, 3), both of which are physiologically available, as the C3 donor and the C5 unit D-ribose 5-phosphate (R5P, 1) as the C3 acceptor to form octulose 8-phosphate (4) (Figure 3), the first sugar intermediate with a C8 skeleton characterized in the lincosamide pathway.21 Mechanistically analogous to known transaldolases, LmbR catalyzes the reaction involving imine bond formation between a conserved L-lysine residue at its active site and the 2-keto group of F6P or S7P, followed by Cα−Cβ bond cleavage via a retro-aldol reaction to produce a C3-unit-conjugated enzyme intermediate, which then reacts D


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are rarely found in natural product biosynthesis and (2) in general, the enzymatic hydroxylation of aromatic rings, including the conversion of L-tyrosine to L-DOPA, is attributed to monophenol monooxygenases that depend on heme (P450 proteins), di-iron, non-heme (pterin, α-ketoglutarate, or copper), or metal-free flavin cofactors.28 The ALDP-related L-tyrosine hydroxylase LmbB2 and its homologues represent a new type of heme peroxidases. They show overall poor sequence similarity with other types but do have a set of motifs/residues characteristic of heme peroxidases that chelate a histidyl-ligated heme ion.26 Consequently, these enzymes could partially follow the catalytic cycle of typical heme peroxidases to form compound I from a ferric resting state (in the presence of L-tyrosine) through compound 0 (Figure 4B). Notably, compound I is also an intermediate in oxygen atom transfer reactions catalyzed by P450 enzymes, which feature a distinct cysteinyl-ligated heme ion.34 Therefore, the subsequent steps are likely reminiscent of those in P450 catalysis: compound I can abstract a hydrogen from L-tyrosine to form hydroxyl Fe(IV) and a substrate radical at the active site, where oxygen rebound leads to ortho-hydroxylation through the formation of an intermediate in complex with the oxygen atom that is coordinated to the Fe(III) center (Figure 4B).

presence of guanosine triphosphate (GTP), to generate guanosine diphosphate (GDP)-D-erythro-D-gluco-octose (8). Further conversion of 8 has yet to be determined biochemically; however, it is reminiscent of the NDPdeoxyaminosugar pathways and likely includes dehydration, transamination, and epimerization to transform 8 to GDP-D-α24 D-lincosamide (9), a key building block for molecular assembly (Figure 3). GDP can serve as a recognition/binding moiety necessary for related enzymes and particularly function as a good leaving group that facilitates transformations proceeding at C1, where the sulfur appendage is added to achieve each end product (Figure 1A). Uncovering the paradigm for lincosamide formation is significant and expands our knowledge of the roles of nucleotide-based activation in the biosynthesis of high-carbon sugars. These unusual sugars have been found as components in an array of microbial natural products and in the lipooligosaccharides of the outer cell walls of Gram-positive bacteria.25 In many cases, the mechanisms of sugar chain elongation, which depend on the manner of C−C bond formation, remain poorly understood.

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PREPARATION OF THE SPECIFIC ALDP RESIDUE PPL Previous isotope-labeling experiments have led to the hypothesis that the formation of various ALDP residues, including the PPL moiety of lincomycin A, involves an oxidation-associated degradation-like process to repurpose seven of L-tyrosine’s nine carbon atoms and the nitrogen atom during the formation of a pyrroline intermediate (Figure 4A).5,12,15,26 This common process has now been enzymatically dissected, as exemplified in the biosynthetic pathway of lincomycin A, including the activities of (1) an L-tyrosine hydroxylase (e.g., LmbB2) that catalyzes an ortho-hydroxylation reaction to produce L-3,4-dihydroxyphenylalanine (L-DOPA, 10);27−29 (2) an L-DOPA 2,3-dioxygenase (e.g., LmbB1) that subsequently conducts oxidative extradiol cleavage for benzoic ring opening and produces a semialdehyde intermediate, which is immediately cyclized to yield an unstable pyrroline product in the imine or enamine form (11 or 11′);27,30,31 and (3) a hydrolase (e.g., LmbA) that is responsible for removing the terminal two-carbon unit of 11 through C−C bond cleavage in the generation of 3-vinyl-2,3-pyrroline-5-carboxylic acid (13) prior to functionalization and diversification to yield individual ALDP members.32 Intriguingly, the enzymatic rearrangement of L-tyrosine through oxidative cleavage during ALDP formation involves so-called “meta-fission” degradation chemistry, which is well-known in the maintenance of the global carbon cycle and is widely employed by aerobic bacteria in catabolizing aromatic rings, either natural or unnatural.33

Non-heme L-DOPA 2,3-Dioxygenase-Catalyzed Oxidative Extradiol Cleavage

The formation of PPL-like ALDP residues involves meta-fission degradative chemistry, as strongly evidenced by the characterized LmbB1 activity, which converts L-DOPA into a yellowcolored pyrroline product through an oxidative-cleavageassociated linearizing process.27,30 This is a process characteristic of non-heme Fe(II)-dependent extradiol dioxygenases that are widely found in catabolic pathways during the degradation of catechol-related compounds.33 Phylogenetically, LmbB1 is a single-domain type I extradiol dioxygenase of the superfamily of vicinal oxygen chelate (VOC)-fold enzymes, which typically utilize Fe(II) and catalyze a reaction involving direct metal ion chelation by vicinal oxygens of the substrate or intermediate and a highly conserved 2-His-1-carboxylate motif in the protein.26 LmbB1 and its homologues appear to be unique in the VOC superfamily and constitute a clade that has poor sequence similarity with other extradiol dioxygenases. Although the intact 2-His-1-carboxylate-like motif characteristic of VOC-fold proteins for metal binding has yet to be determined,30 LmbB1-mediated oxidative cleavage during L-DOPA 2,3oxygenation may proceed in a manner similar to that mediated by characterized non-heme Fe(II)-dependent extradiol dioxygenases (Figure 4B). The catechol portion of L-DOPA may bind to ferrous ion in a bidentate manner as a monoanion. After binding with the oxygen molecule, transfer of an electron from the substrate to O2 can occur, resulting in the formation of a semiquinone−Fe(II)−superoxide species, which could then undergo radical coupling to produce an iron−alkylperoxo intermediate. A ring expansion might occur through alkenyl migration, Criegee-like rearrangement, and subsequent O−O bond cleavage, followed by hydrolysis of the resulting lactone by Fe(II)-bound hydroxide to yield a linear semialdehyde intermediate. Most likely, the cyclization of this intermediate to form the pyrroline product 11 is enzyme-independent and proceeds spontaneously.31

Heme Peroxidase-Catalyzed Ortho Hydroxylation of L-Tyrosine

The characterization of LmbB2 and its counterpart Orf13 in the biosynthesis of the PBD member anthramycin revealed the Ltyrosine hydroxylase in ALDP formation as a heme-dependent peroxidase that catalyzes ortho-hydroxylation in the presence of hydrogen peroxide.28,29 Intriguingly, Orf13 activity, which requires the cofactor heme b in the coordination of a histidyl-ligated metal ion, is sensitive to hydrogen peroxide, and thus, preincubation of the enzyme with L-tyrosine is necessary for L-DOPA production. Peroxidase activity for orthohydroxylation is unusual because (1) peroxidases, which are widely present in degradation and H2O2-scavenging pathways, E


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Figure 5. Proposed mechanism of γ-GT homologue-catalyzed C−C bond cleavage.

γ-Glutamyltranspeptidase Homologue-Catalyzed C−C Bond Cleavage

glycine tripeptide, or from GSH S-conjugates during antioxidant defense, detoxification, and inflammatory processes.36 γ-GTs belong to the superfamily of N-terminal nucleophile (Ntn)-hydrolase fold proteins and exhibit activity for autocatalytic cleavage of a peptide precursor to form a functional heterodimer, in which a newly released N-terminal residue (e.g., typically L-threonine, L-serine, or L-cysteine) acts as a nucleophile and mediates amide bond hydrolysis through the formation of an acyl-nucleophile enzyme intermediate.37 γGT homologues are widely present in living organisms, including Gram-positive actinobacteria; however, these bacteria do not involve GSH metabolism.38 Actinobacteria include antibiotic-producing actinomycetes, such as the producers of the ALDP-containing metabolites discussed here, and the dominant thiols in these microorganisms are mycothiol (MSH), a cysteinyl pseudodisaccharide, and ergothioneine (EGT), a histidine betaine derivative,39,40 neither of which contains γ-glutamyl, thereby arguing against the role of LmbA and its homologues as γ-GTs for γ-glutamyl transfer or hydrolysis in actinobacteria. Mechanistically, the transformation of 11 to 13 in the formation of ALDP residues is reminiscent of the γ-GT-associated hydrolytic process and likely requires nucleophilic attack by water onto the α-keto group of 11 to produce oxalic acid (12) as a coproduct.

The identification of LmbA and its homologues (e.g., Orf6 in anthramycin biosynthesis) as unusual hydrolases for C−C bond cleavage benefited from analyzing related S. lincolnensis mutants in PPL production and, particularly, from comparing the biogenesis of ALDP residues and determining a hydrolytic mechanism from the reaction.32,35 A sequence alignment of all available biosynthetic gene clusters of ALDP-containing metabolites revealed four conserved genes, as exemplified by lmbB1, lmbB2, lmbY, and lmbA in the lmb cluster, and their deduced proteins were thus assigned for common reactions that occur during the formation of ALDP residues. LmbB2 and LmbB1 are known to form an enzyme pair that is responsible for the first two steps in the transformation of L-tyrosine to 11. LmbY appears to be a coenzyme F420-dependent oxidoreductase and is believed to function at a later stage for olefinic double-bond reduction,15,16 thereby leaving LmbA as the only candidate to catalyze the next shared reaction for removal of the terminal two-carbon unit. LmbA is homologous to various γ-glutamyltranspeptidases (γ-GTs), which are ubiquitous enzymes that catalyze the transfer or hydrolysis of γ-glutamyl, often from the smallmolecule thiol glutathione (GSH), a γ-glutamyl-L-cysteinyl-LF


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Figure 6. EGT- and MSH-mediated functionalization of the C8 aminosugar unit.

Indeed, LmbA-like γ-GT homologues share an autoproteolytic activity for self-activation, which leads to unprecedented Ntn-hydrolase activity for C−C bond cleavage (Figure 5).32 Autoproteolysis occurs specifically within the highly conserved DT motif of a precursor peptide, where L-threonine acts as an internal nucleophile to mediate hydrolysis of the preceding amide bond of the protein. This process results in an active heterodimer composed of a C-terminal Asp-containing large subunit and an N-terminal Thr-containing small subunit. The newly released L-threonine residue then functions as an Nterminal nucleophile at the active site to form an acyl-Thr enzyme intermediate for the subsequent acyl hydrolysis reaction. Unlike known Ntn-hydrolase fold proteins (including γ-GTs) that are essentially amidohydrolases, LmbA and its homologues target the carbonyl group of the carbon side chain of 11 and catalyze C−C bond cleavage to yield the unstable enamine diene 13 and an oxalyl-Thr enzyme intermediate, the latter of which is subsequently hydrolyzed to release oxalic acid. Interestingly, in addition to lmbA, which encodes a pathwayspecific γ-GT, S. lincolnensis harbors lmbA2991, which is not part of the lmb cluster and codes for a non-pathway-specific γGT that can partially compensate for the loss of lmbA in the formation of the PPL moiety of lincomycin A.32 Notably, there are a number of γ-GT homologues that have initially been annotated as γ-GTs in Gram-positive actinobacteria, where GSH metabolism (often including detoxification processes) is not involved.38−40 Unveiling their functions should be extremely interesting because Ntn-hydrolase activities could be associated with new biochemical reactions.

and appears to rapidly undergo tautomerization to an imine,32 which is resistant to LmbW activity and cannot be Cmethylated. A lack of LmbW activity can lead to the formation of a trans-4-ethyl-L-proline residue and, eventually, the production of lincomycin B after molecular assembly; lincomycin B is a naturally occurring analogue of lincomycin A in S. lincolnensis (Figure 1A).41 MSH- and EGT-Programmed C8 Sugar Functionalization

During the biosynthesis of lincosamide antibiotics, the functionalization of the lincosamide unit includes an assembly with PPL (for lincomycin A) or L-proline (for celesticetin and Bu2545) and replacement of the α-O-linked GDP with a variable α-S-linked appendage (Figure 1A). This functionalization process was poorly understood until an unprecedented constructive role of two small-molecule thiols, MSH and EGT, was uncovered.24 Small-molecule thiols are a class of organosulfur compounds that are well-known for their redoxassociated protective role.38 MSH and EGT,39,40 which are functionally equivalent to GSH in eukaryotes and Gramnegative bacteria, are dominant thiols in Gram-positive actinomycetes and function as reductive cofactors, maintain intracellular redox homeostasis, and detoxify cells by scavenging alkylating agents, free radicals, and xenobiotics. Remarkably, these thiols can function beyond a role in cell protection and can program the lincosamide functionalization process through two unusual S-glycosylations (Figure 6).24 EGT mediates molecular assembly as a cryptic carrier, thereby providing the first paradigm for EGT-dependent biochemical reactions. In contrast, MSH is a sulfur donor after thiol exchange, representing a new MSH-associated paradigm necessary for the incorporation of sulfur, an element that is ubiquitous in living systems and essential for life.

Building-Up Process in ALDP Formation

Building-up reactions toward the formation of various ALDP residues start with the functionalization and diversification of 13.15 Specifically, in the biosynthetic pathway of lincomycin A, these reactions could include LmbW-catalyzed C-methylation, LmbY-catalyzed hydrogenation, and LmbX-catalyzed epimerization to achieve PPL (Figure 4A).35,41 C-Methylation immediately follows LmbA-catalyzed C−C bond cleavage because enamine 13 has been shown to be extremely unstable

Common Route for C8 Sugar Transfer and Modification

The first S-glycosyltransferase (e.g., LmbT in lincomycin A biosynthesis) acts on GDP-D-α-D-lincosamide (9) and catalyzes a reversible SN2 displacement to transfer the C8 sugar unit from GDP via a β-S-linkage onto the thiol EGT, yielding 16, an EGT S-conjugated lincosamide (Figure 6).27 Incorporation with PPL G


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Figure 7. Difference in the formation of lincomycin A (red) and celesticetin (purple). (A) Biosynthetic pathways. (B) Proposed mechanisms of LmbF-mediated β-elimination for C−S bond cleavage and CcbF-mediated decarboxylation-coupled oxidative deamination.

ase (e.g., LmbV), 24 which functions in a nucleotideindependent manner and belongs to the DinB-2 superfamily, including over ten thousand members with activities related to various small-molecule thiols such as GST and MSH.43 The members of this superfamily contain a conserved DinB-2-like thiol-binding domain in combination with variable functional domains, indicating that they participate in different thiolinvolved biochemical processes. Intriguingly, formation and processing of 18 (e.g., by the Mca-like amidase LmbE) are mechanistically analogous to a typical MSH-mediated detoxification pathway, which relies on conjugation of MSH to an electrophilic toxin and subsequent hydrolysis of the resulting MSH S-conjugate (Figure 6).44 The similar pathway leads to the production of a pseudodisaccharide unit, 1-O-glucosamineD-myo-inositol (GlcN-Ins, for MSH regeneration), and a mercapturic acid derivative, 19, which is structurally related to excretive N′-acetyl-L-cysteinyl products found in GSH- or MSH-mediated detoxification metabolism.

then occurs in a manner similar to that catalyzed by wellestablished nonribosomal peptide synthetases (NRPSs).24,42 Specifically, a discrete adenylation protein (e.g., LmbC) activates PPL with adenosine triphosphate (ATP) and transfers it onto a 4′-phosphopantetheinylated thiolation (T) domain (e.g., the N-terminal domain of the bifunctional protein LmbN), and a discrete condensation (C) protein (e.g., LmbD) catalyzes the transpeptidation between activated PPL and 16 to form a C−N linkage and produce the EGT Sconjugated intermediate 17. The condensation reaction likely requires EGT as a necessary recognition/binding moiety because it cannot proceed between PPL and GDP-activated 9. In intermediate 17, the C8 sugar unit is attached to EGT via a β-S-linkage. EGT is a good leaving group and thus facilitates nucleophilic attack by MSH at C1 in the production of 18, an MSH S-conjugated intermediate that shares an α-S-linkage with mature lincosamide antibiotics (Figure 6). The thiol exchange between EGT and MSH proceeds through an irreversible SN2 displacement and is catalyzed by the second S-glycosyltransferH


Accounts of Chemical Research Diverse Maturation of Lincosamide Antibiotics

Following N′-deacetylation of 19 to give 20 and subsequent Nmethylation of the PPL or L-proline moiety to yield L-cysteinyl S-conjugate 21 (e.g., catalyzed by LmbJ),35,45 the downstream route for maturation of lincosamide antibiotics focuses on differential processing of the remaining L-cysteinyl group (Figure 7A). This processing is a branching point in the biosynthesis of lincosamide antibiotics and primarily depends on the activity of a pyridoxal-5′-phosphate (PLP)-dependent protein, i.e., LmbF in lincomycin A biosynthesis or CcbF in celesticetin biosynthesis,46−48 two proteins that are phylogenetically related but functionally distinct. PLP-dependent proteins participate in numerous biochemical processes through a wide variety of reactions that occur on substrates bearing an amino group, including racemization, decarboxylation, transamination, retro-aldol cleavage, and Claisen condensation.49 All of these reactions depend on PLP chemistry through the formation of a common aldimine intermediate but diversify after this step as a result of minute differences in the active sites of the enzymes. LmbF is responsible for β-elimination, yielding thiol intermediate 22 through C−S bond cleavage (Figure 7B).46−48 This intermediate is highly reactive and immediately undergoes LmbG-catalyzed S-methylation to produce the end product with a methylmercapto group. Notably, the conversion of mercapturate derivatives (e.g., 19) to excretive thiomethyl products (e.g., 22) was previously established as an extended detoxification pathway in thiol-mediated metabolism.44 In contrast, CcbF catalyzes an unusual conversion that involves decarboxylation-coupled oxidative deamination of the Lcysteinyl group (Figure 7A),47,48 yielding aldehyde intermediate 23, which then undergoes Ccb5-catalyzed hydrogenation to form a two-carbon alcohol linker and Ccb4-catalyzed Omethylation during the production of desalicetin, a nonsalicyclic precursor of celesticetin in S. caelestis. In the presence of O2, a radical semiquinone intermediate and superoxide anion could be formed via single electron transfer and then be recombined to produce a hydroperoxyl aldimine, which is likely subjected to H2O2 elimination and subsequent imine hydrolysis to yield an aldehyde (Figure 7B).48 PLP chemistry is common for carbanion stabilization; however, its association with O2 activation and incorporation in catalysis has rarely been described.50 Remarkably, LmbF-catalyzed β-elimination and CcbF-catalyzed decarboxylation-coupled oxidative deamination are exchangeable between the biosynthetic pathways of lincomycin A and celesticetin,48 thereby allowing for in vitro combinatorial biosynthesis of several hybrid lincosamide antibiotics, including Bu-2545 (Figure 1A), whose biogenesis remains to be determined in Streptomyces sp. H230-5. Conversion of desalicetin to celesticetin requires the activities of the acylCoA ligase Ccb2 and the acyl transferase Ccb1, which are responsible for functionalizing the two-carbon alcohol linker using a salicyclic moiety.51 Both Ccb2 and Ccb1 appear to be promiscuous in substrate tolerance, and their combination/ permutation with proteins related to celesticetin biosynthesis (i.e., CcbF, Ccb4, and Ccb5) in treatment of lincomycin intermediate 20 or 21 further developed the in vitro combinatorial biosynthesis method. This led to the construction of a lincosamide antibiotic-like natural product library comprising over 150 hybrid members.51

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CONCLUSION

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AUTHOR INFORMATION

Natural-product-mediated communication plays a crucial role in the interface between living organisms and their environments. The evolution of active small molecules in nature appears to occur over a continuous spectrum spanning millennia, and the imaginable permutations or mutations of prototypes, which could involve biosynthetic pathways, biochemical reactions, catalytic enzymes and encoding genes, create natural products that display an unimaginable diversity of chemical structures and biological functions. To date, substantial unprecedented biosynthetic paradigms have been established, including that of lincosamide antibiotics, allowing the application of the generality and specificity in biosynthesis to account for small-molecule development in nature. We have now witnessed the relevance of distinct paradigms for the biosynthesis of almost all natural product families. However, biosynthetic machineries that involve many reactions typically associated with degradation and detoxification in the creation of active small molecules, such as lincosamide antibiotics, are uncommon and could arise from evolution through the intersection of anabolic and catabolic chemistry. The paradigm for lincosamide antibiotic biosynthesis includes unique enzymes, such as the DinB-2 fold protein for thiol exchange, the γ-GT homologue for C−C bond cleavage, and the PLPdependent protein for diverse S-functionalization, generating interest in how nature has developed stunningly diverse biological functions using a limited range of protein scaffolds. A greater understanding of the related biochemical processes would facilitate the design and development of comparable biosynthetic machineries for expansion of the molecular diversity.

Corresponding Author

*E-mail: [email protected]. Tel: 86-21-54925111. Fax: 86-2164166128. ORCID

Wen Liu: 0000-0001-8835-8012 Author Contributions §

D.Z. and Z.T. contributed equally to this work.

Notes

The authors declare no competing financial interest. Biographies Daozhong Zhang received his B.S. in Chemistry at Ocean University in China in 2011. In 2017, he obtained his Ph.D. under the supervision of Professor Wen Liu at the Shanghai Institute of Organic Chemistry (SIOC), Chinese Academy of Sciences (CAS). He is currently working on natural product-related enzymology as a research associate. Zhijun Tang graduated with a B.S. from Sichuan University in China in 2014. He is currently pursuing his Ph.D. studies at SIOC, CAS, under the supervision of Prof. Wen Liu. His research interests include natural product-related genome mining and enzymology studies. Wen Liu obtained his Ph.D. from the Chinese Academy of Medical Sciences in 2000. After postdoctoral studies at UC-Davis (2000− 2001) and UW-Madison (2001−2003), he joined SIOC, CAS, in 2003. His research interest focuses on natural product biosynthesis (genetics, biochemistry, and chemistry), engineering, and genome/ transcriptome mining. I


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ACKNOWLEDGMENTS This work was supported in part by grants from NNSFC (21520102004, 21472231, 31430005, 21750004, and 21621002), CAS (QYZDJ-SSW-SLH037 and XDB20020200), STCSM (17JC1405100 and 15JC1400400), MST (2018ZX091711001-006-010), and the K. C. Wang Education Foundation.

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