Substrate-Controlled Stereochemistry in Natural Product Biosynthesis

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Substrate-controlled stereochemistry in natural product biosynthesis Wei Ding, Yongzhen Li, and Qi Zhang ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 06 Apr 2015 Downloaded from http://pubs.acs.org on April 7, 2015

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ACS Chemical Biology

Substrate-controlled stereochemistry in natural product biosynthesis Wei Ding, Yongzhen Li, and Qi Zhang* Department of Chemistry, Fudan University, Shanghai, China, 200433

Table of Contents

Keywords 1. RiPP: ribosomally synthesized and posttranslationally modified peptide 2. Lanthipeptide: lanthionine-containing peptide 3. Sactipeptide: sactionine-containing peptide 4. Radical SAM: Radical-based enzymatic reactions initiated by reductive cleavage of the C-S bond of S-adenosylmethionine 5. SPASM: Subtilosin/PQQ/Anaerobic Sulfatase Maturating enzymes, a subgroup of radical SAM proteins containing one or more iron-sulfur clusters in addition to the one required for SAM cleavage. 6. SAC: substrate-assisted catalysis, a process in which a functional group in a substrate contributes to the catalysis 7. ACP: acyl carrier protein 8. HR-PKS: Highly reducing polyketide synthase 1

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Abstract Enzymes are generally believed to be highly regio- and stereo-selective catalysts that strictly control the reaction coordinates and dominate the final catalytic outcomes. However, recent studies have started to suggest that substrates sometimes play key roles in determining the product selectivity in enzyme catalysis. Here we highlighted several enzymatic reactions in natural product biosynthesis, in which the stereoselectivity is, at least in large part, governed by the intrinsic properties of the substrate rather than enzyme characteristics. Understanding the mechanism of the substrate-controlled stereospecificity may not only expand our knowledge of enzyme catalysis and enzyme evolution, but also guide bio-engineering efforts to produce novel valuable products.

Introduction Enzymes are macromolecular bio-catalysts responsible for millions of metabolic reactions that sustain life. Through billions of years of evolution in life history (1), these catalysts have evolved to be highly specific and efficient, which not only tremendously speed up reaction rates but also enforce strict regio- and stereo-specificity in the product formation. Except for a small fraction of enzymes that are RNA-based molecules (ribozymes) (2), most enzymes are linear chains of amino acids folded into three dimensional scaffolds. The well-organized microenvironments provided by the enzymes fold their substrates into reactive conformations and orient the breaking and formation of chemical bonds in a highly regio- and stereoselective manner. Accordingly, it is generally believed that enzymes 2


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strictly control the reaction coordinates and govern the final outcomes of catalysis, producing the desired products necessary for life.

While enzyme-controlled selectivity likely applies to many biochemical reactions, recent evidence has started to suggest that substrates sometimes play key roles in determining the results in enzyme catalysis. In these reactions, the final products are, at least in large part, governed by the intrinsic property of the substrate rather than enzyme characteristics. A remarkable observation is the ability of the same enzyme to generate different stereochemical outcomes for different physiological substrates and/or different sites of the same substrate. It has been well-recognized that enzyme chemistry could be altered and manipulated by using mechanism-based probes and inhibitors (3). Substrate-assisted catalysis, a process in which a functional group in a substrate contributes to the catalysis, has also been demonstrated for several enzymes (4). However, the fact that an enzyme catalyzes the same type of reaction on similar natural substrates via the same mechanism but generates distinct stereochemical outcomes is intriguing. In this review, we highlight several reactions in which the stereochemistry seems to be largely controlled by the chemical properties of the substrates and can be reversed when different physiological substrates are used. These examples not only demonstrate the intriguing enzymology that constitute fascinating puzzles for aficionados of enzyme stereochemistry, but also shed interesting lights on the evolution of enzyme catalysis.

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Lanthipeptide biosynthesis Lanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptides characterized by the presence of the thioether crosslinked amino acids lanthionine (Lan) and methyllanthionine (MeLan) (Figure 1A) (5-9). These peptide natural products are widely distributed in taxonomically distant species and display very diverse biological activities, ranging from antimicrobial to antiallodynic (10-12). Lanthipeptides are generated from a ribosomally synthesized linear precursor peptide (generically termed LanA) and therefore belong to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs) (13). The precursor peptide LanA consists of an N-terminal leader peptide that is essential for recognition by posttranslational modifying enzymes, and a C-terminal core peptide where all posttranslational modifications take place (14-16). Biosynthesis of Lan and MeLan rings is achieved by the initial dehydration of Ser and Thr residues in the precursor peptides that produces dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively. This step is followed by stereoselective intramolecular Michael-type addition of Cys thiols to the newly formed dehydroamino acids (Figure 1A). In some cases, this reaction is coupled with a second Michael-type addition of the resulting enolate to a second Dha to produce a labionin structure (17). Four classes of biosynthetic enzymes are known to catalyze lanthionine formation (6, 18). These enzymes differ in structure and organization but all contain a cyclase, which exists either as a stand-alone protein (generically termed LanC) in class I lanthipeptide synthetases, or as a LanC-like cyclase domain that is part of a multi-functional protein 4


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in class II, III, and IV lanthipeptide biosynthesis (class II lanthipeptide synthetases are generically termed LanM). The lanthipeptide ring systems resulted from the action of LanC/LanC-like enzymes range from simple nonoverlapping rings to very complex, intertwined rings (5, 6), highlighting the tremendous structural diversity of lanthipeptides. More intriguingly, many lanthipeptide synthetases, including prochlorosin synthetase ProcM (19) and several other cyanobacterial LanMs (20), produce (Me)Lan structures on a series of precursor peptides with highly diverse sequences. The active sites of these enzymes need to accommodate a set of structurally highly diverse substrates while at the same time strictly control the regioand stereo-selectivity in the multiple reaction steps. The manner by which these impressive processes are achieved remains largely enigmatic at present.

The first evidence of substrate-controlled selectivity in lanthipeptide biosynthesis came from a phylogenetic study, which shows that enzymes producing structurally similar lanthipeptides sometimes fall into different phylogenetic clades, whereas phylogenetically closely related enzymes generate products with distinct rings (18, 21). This observation suggests that the ring topology of the final lanthipeptides may be determined mainly by the sequence of the precursor peptides rather than by the enzymes. The fact that ericin S and ericin A possess very different C-terminal ring topologies but are produced by the same biosynthetic enzymes is consistent with this hypothesis (22). This hypothesis was further supported by studies with two chimeric peptides, showing that the nisin and prochlorosin biosynthetic enzymes can produce 5



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the correct ring structures of epilancin 15X and lacticin 481, respectively (Figure 1B) (18). The ring structure of lacticin 481 shares no similarity with those of prochlorosins (which consist of thirty members), suggesting that the precursor peptide substrate determines the site selectivity of cyclization whereas the cyclase does not appear to enforce the rings to be formed. However, cyclases are still indispensable for the process, as non-enzymatic cyclization of dehydrated substrates at increased pH either did not yield the correct structure (23) or is far slower than the enzymatic process (24).

An even more compelling piece of evidence of substrate-controlled selectivity in lanthipeptide biosynthesis comes from the study of (Me)Lan stereochemistry (25, 26). Until recently it was generally believed that LanC/LanC-like cyclase-catalyzed intramolecular addition of the thiol of Cys to Dha and Dhb always produced (2S, 6R)-Lan (hereafter termed DL-Lan) and (2S, 3S, 6R)-MeLan (hereafter DL-MeLan), respectively (Figure 1A). However, structural analysis of the two-component lanthipeptides cytolysin L and cytolysin S showed that both peptides contain (2R, 3R, 6R)-MeLan (LL-MeLan) besides DL-Lan, and cytolysin L also contains (2R, 6R)-Lan (LL-Lan) (Figure 1A) (25). Cytolysin L and S are produced from the precursor peptide CylLL and CylLS, respectively, by a single class II lanthipeptide synthetase CylM (27). Two types of (Me)Lan structures are therefore produced by a single enzyme on a single polypeptide substrate.

6


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The LL-Lan and LL-MeLan in cytolysin are all formed from an S/T-S/T-X-X-C motif, in which X represents amino acids other than Ser, Thr and Cys, and both S/T residues are dehydrated (25). This observation suggests that the S/T-S/T-X-X-C motif likely induces the formation of the unusual LL stereochemistry of (Me)Lan. Notably, several documented lanthipeptides, including geobacillin II (28), Smb (29), haloduracin (30), and carnolysin (31), contain such S/T-S/T-X-X-C motif, and the presence of LL-(Me)Lan in the latter two lanthipeptides was further experimentally validated (25, 31), pointing out a sequence-controlled stereochemistry in lanthionine biosynthesis. Indeed, LL-MeLan residues were also generated from various chimeric peptides that contain the CylLS core peptide and different LanA leader peptides, by using the corresponding lanthipeptide synthetases (Figure 1C) (26). These synthetases include HalM2, LtnA2, and ProcM, the latter two enzymes do not produce LL-Lan and LL-MeLan in their natural substrates, and hence are unlikely to have evolved the ability to form LL-MeLan. Again, it is noteworthy that no sequence similarities are shared between cytolysin and prochlorosins. Furthermore, ProcM is phylogenetically very different from CylM and HalM2 and contains a distinct set of ligands for binding of an active site Zn2+ ion (18), which is believed to be important for catalysing the cyclization reaction (32, 33). Therefore, it appears to be the substrate sequence that mainly determines the stereochemical outcome in lanthionine biosynthesis rather than the enzymes.

Interestingly, the LL stereochemistry of MeLan could be completely reversed to DL 7



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by simply replacing the second Thr in the T-T-X-X-C motif to Ala, strongly suggesting that the second Dhb residue plays a pivotal role in LL-MeLan formation (Figure 1C) (26). Computational studies for three oligopeptides showed that LL-MeLan formation is kinetically favored for Dhb-Dhb-Pro-Ala-Cys and Dhb-Dhb-Trp-Pro-Cys, the two sequences from cytolysin S and haloduracin β, respectively.

On

the

other

hand,

DL-MeLan

formation

is

favored

for

Dhb-Ala-Trp-Pro-Cys (26). These analyses reproduced the experimentally observed selectivity in lanthionine formation. Undoubtedly, three-dimensional structural information, particularly structures with a substrate bound to the enzyme, will be necessary to evaluate the computational results and provide further insights into these intriguing catalytic processes.

The substrate-controlled selectivity in lanthipeptide biosynthesis expands our understandings of enzyme mechanism and paves the way for future studies on lanthipeptide-related natural products. Although thus far, the LL stereochemistry is only associated with the S/T-S/T-X-X-C motif, it might be possible that other unknown sequences could also generate LL-(Me)Lan or even favor other diastereomers if syn addition can be achieved. This hypothesis is tempting because lanthipeptides are widespread in nature with vast sequence diversity (18, 34, 35), and thus far only a very small number of them have been subjected to stereochemical analysis. Also interesting is the question whether labionin, which to date has only been found with a (2R, 4S, 8R)-configuration (17), could exist in different 8


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stereochemical

configurations.

Understanding

the

substrate-controlled

stereochemistry in lanthipeptide biosynthesis might also allow engineering of novel lanthipeptide scaffolds with desired biological activities.

Sactipeptide biosynthesis Sactipeptides are a small but growing family of RiPPs containing intramolecular thioether linkages between cysteine sulfur and the α-carbon of another amino acid (Figure 2A) (13, 36). Thus far six members of this family have been reported, including subtilosin A (37, 38), subtilosin A1 (a subtilosin A T6I mutant) (39), thurincin H (40, 41), the cannibalistic sporulation killing factor (SKF) (42), and the anticlostridial thuricin CD that consists of two peptides Trn-α and Trn-β (43, 44) (Figure 2B). The key sulfur to α-carbon crosslinks in sactipeptides are produced by radical S-adenosylmethionine (SAM) enzymes (we here propose the name sactionine to describe the amino acids containing Cys sulfur to α-carbon thioether linkages, in a way

similar

to

lanthionine;

accordingly,

sactipeptides

are

defined

as

sactionine-containing peptides) (Figure 2A). The sactionine-synthesizing radical SAM enzymes (sactionine synthetases) contain two [4Fe-4S] clusters (36, 45, 46) and belong to the SPASM (Subtilosin/PQQ/Anaerobic Sulfatase Maturating enzymes) family (47-50). The N-terminal cluster (radical SAM cluster) binds and reductively cleaves SAM to produce a highly reactive 5ˊ-deoxyadenosyl radical that initiates the reaction (51-53). The C-terminal [4Fe-4S] cluster (auxiliary cluster) has been proposed to bind the Cys thiol groups of the precursor peptide to facilitate the 9



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radical-based thioether linkage (36, 45), in a fashion similar to the catalysis of isopenicillin N synthase (54). It should be noted that thiopeptide cyclothiazomycin also contains a sactionine residue (55-57), which apparently is not created via radical SAM chemistry (58, 59); this compound is not regarded as a sactipeptide.

Like lanthipeptides, some sactipeptides contain sactionine residues with different configurations at the α-carbon stereocenter in one peptide. Whereas thuricin H only contains (2S)-sactionine (hereafter D-Sac) (41) and SKF only contains one sactionine for which the stereochemistry is yet to be determined (42), the other three known sactipeptides have both (2R)-sactionine (hereafter L-Sac) and D-Sac (Figure 2B). Neither an epimerase nor a putative epimerase/isomerase candidate protein is encoded in the gene clusters (36), suggesting that the two types of stereocenters are generated by the same sactionine synthetase. Therefore the stereochemical outcome is likely governed by the spatial orientation of the substrates, in a way similar to lanthipeptide biosynthesis discussed above.

To date no structural information is available for sactionine synthetases, and the stereochemical reaction process in sactipeptides biosynthesis is largely unknown. It has been shown that lipoyl synthase (LipA) contains a deep channel between the two [4Fe-4S] clusters for substrate binding (60). Similarly, production of D-Sac and L-Sac may occur in a binding channel between the two [4Fe-4S] clusters of a sactionine synthase. A hypothetic scenario would be that thioether bond formation from the 10


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α-carbon radical intermediate occurs from the opposite side of α-hydrogen abstraction by the 5ˊ-deoxyadenosyl (dAdo) radical (Figure 3). Accordingly, formation of a D-Sac may possible be kinetically favored over formation of an L-Sac, because the latter would involve structural re-orientation of the α-carbon radical intermediate (Figure 3). We noted that sactionine generated from the first N-terminal Cys always has a D-configuration (Figure 2B). A possible explanation is that the thioether bond formation mostly starts from the first N-terminal Cys, and the first thioether linkage produced on a linear, flexible peptide substrate may always adopt a kinetically favored D-configuration (Figure 3). Formation of the thioether crosslinks renders the peptide structure more rigid and enforces different stereochemistry to be generated in the following thioether bond linkage. Further studies, both on characterization of novel sactipeptides that likely are widespread in nature (61, 62), and on detailed catalytic mechanism of sactionine synthetases, are required for unveiling the detailed mechanism of the stereochemical control process in sactipeptide biosynthesis.

Ketoreduction in hypothemycin biosynthesis Polyketides represent a large group of natural products with highly diverse structures and biological activities, and constitute an important source for pharmaceuticals. The carbon backbones of polyketides arise from the programmed assembly of short chain acyl coenzyme A (CoA) precursors in a way similar to fatty acid biosynthesis (63-65). In fatty acid biosynthesis, the β-keto group of the nascent polyketone chain is fully processed by stepwise ketoreductase (KR)-, dehydratase (DH)-, and enoyl reductase 11



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(ER)-catalyzed reactions to yield a saturated acyl chain (66). In contrast, polyketide synthetases (PKSs) use optional reductive steps to process the β-keto group, thus giving rise to a more complex pattern of functionalities. PKSs and fatty acid synthetases (FASs) have been classified into different types according to their enzyme architectures and catalytic mechanisms. Type I systems are multifunctional proteins consisting of linearly arranged and covalently fused domains for individual catalytic activities that are organized in a modular manner (67-69), whereas type II systems are dissociable multienzyme complexes consisting of discrete and usually monofunctional proteins (70).

The first step in β-keto processing in polyketide biosynthesis is catalyzed by the KR domain, which belongs to the short chain dehydrogenase/reductase superfamily, and produces an alcohol of L- or D- stereochemistry using NADPH (71). KRs from bacterial type I PKSs that produce L- or D-alcohols are termed type A and type B KRs, respectively, which are readily recognized by their characteristic amino acid sequence fingerprints (72-75); these sequence fingerprints, however, may not fully apply to fungal PKS KRs (76). Although low stereospecificity has in some cases been observed for KR-catalyzed reactions with unnatural substrates (77, 78), ketoreduction is typically highly stereospecific with the native acyl carrier protein (ACP)-bound substrate, involving the stereospecific transfer of the 4-pro-S hydride from NADPH to the ketone of the substrate (79, 80). Structural studies have suggested that the enzyme active site residues guide the entrance of substrates and fix the β-keto group relative to 12


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NADPH, facilitating the stereospecific hydride transfer (72, 81, 82).

The stringent KR stereospecificity, however, has recently been demonstrated to be tunable by the length of the nascent polyketide chain in some cases such as the biosynthesis of the fungal polyketide hypothemycin (76). Hypothemycin is a resorcylic acid lactone biosynthesized by the sequential action of a highly reducing PKSs (HR-PKS) Hpm8 and a nonreducing PKS (NR-PKS) Hpm3 (Figure 4A) (83, 84). HR-PKSs from filamentous fungi are among the most complex and intriguing type I PKSs (67, 68, 85-88), which use the same protein domains repeatedly for each stage of chain growth and produce a diverse array of polyketides including the cholesterol-lowering drug lovastatin (89). Hpm8 and Hpm3 work sequentially and produce an intermediate (6ˊS, 10ˊS)-7ˊ,8ˊ-dehydrozearalenol (DHZ) that is subsequently tailored to hypothemycin (Figure 4A). The two chiral centers at C6ˊ and C10ˊ in DHZ suggest that the KR in Hpm8 performs unusual opposite stereochemistries in ketoreduction. In vitro study of Hpm8 KR with a series of synthetic acyl-tethered S-N-acetyl cysteamine (S-NAC) compounds confirmed this proposal,

showing

that

the

triketide-,

tetraketide-,

pentaketide-,

and

hexaketide-S-NAC substrates were all exclusively reduced to the corresponding D-alcohols (Figure 4B). On the other hand, the diketide substrate acetoacetyl-S-NAC was predominantly (more than 91% stereoselectivity) reduced to an L-alcohol (Figure 4B). These results clearly demonstrate the substrate-controlled stereospecificity of Hpm8 KR, which normally performs D-type reduction but could also perform L-type 13



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reduction with a short, diketide substrate.

The substrate-controlled stereochemistry of Hpm8 KR is different from those in lanthipeptide and sactipeptides biosynthesis. In lanthipeptide biosynthesis, the T-T-X-X-C substrate was shown both experimentally and computationally to have an intrinsic preference for LL-MeLan formation (26). In contrary, the β-ketoacyl thioester substrates of Hpm8 KR apparently have no preference for either D- or L-type reduction, indicating the substrate length-tuned stereochemistry is likely a special characteristic of Hpm8 KR and may not apply to other HR-PKS KRs. Indeed, Rdc5, a HR-PKS that exhibits a head-to-tail sequence homology with Hpm8 over their entire length and produces a biosynthetic intermediate of radicicol (monocillin II) possessing a similar carbon scaffold as that of DHZ (Figure 4C) (76), reduces the same diketide intermediate with the opposite stereochemistry to Hpm8 to produce a D-alcohol (Figure 4D, equation i). Intriguingly, the stereochemistry of Hpm8 KR could be reversed by swapping part of its internal sequences for the corresponding sequences of the Rdc5 KR (Figure 4D, equation ii), allowing for the biosynthesis of a novel unnatural diastereomer of DHZ (epi-DHZ) (Figure 4E) (76). These results highlight the intriguing biochemistries of HR-PKSs and set the stage for fully deciphering the complex programming rules of these enigmatic megaenzymes.

Acyl carrier protein (ACP)-directed stereochemistry ACPs are small individual proteins in type II FAS and PKS systems or discrete folded 14


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domains in the multi-domain type I systems, and are characterized by a fold consisting of three major α-helices between 60–100 amino acids (90-93). The protein is expressed in an inactive apo form, and a 4'-phosphopantetheine moiety is post-translationally attached to a conserved serine residue to afford the active holo form. ACPs sequester the growing chains in an interior hydrophobic cavity that protects these intermediates from non-selective reactivity (90-93). The flexible, 18 Å phosphopantetheine arm delivers the covalently tethered intermediates to spatially distinct catalytic partners, thereby increasing the effective molarity of the intermediate and allowing an assembly line-like process to be carried out.

The β-hydroxyl group resulting from ketoreduction of the growing polyketone chain can be eliminated by a DH domain. These dehydratases catalyze β-elimination in a strict syn manner (94). Three stereochemically-characterized modular PKS DH domains, including EryDH4 from the erythromycin synthase (95), NanDH2 from the nanchangmycin synthase (96), and TylDH2 from the tylactone synthase (75), have been

shown

to

display

strict

(2R,3R)-2-methyl-3-hydroxyacyl-ACP stereospecific

specificity substrates

dehydration

syn

to

for and

give

the

diastereomeric

catalyze the

completely corresponding

trans-(E)-2-methyl-2-enoyl-ACP products. RifDH10, the dehydratase domain from the terminal module of the rifamycin polyketide synthase, shows high sequence similarity with EryDH4, NanDH2, and TylDH2. Indeed, as anticipated RifDH10 performed

stereospecific

catalysis

on

thioester

substrates

15


containing

the


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Page 16 of 32

(2R,3R)-2-methyl-3-hydroxypentanoyl group tethered to pantetheine, N-acetyl cysteamine (NAC) (Figure 5A), or EryACP6 (a heterologous ACP from erythromycin synthase)

(Figure

5B)

(97).

However,

when

presented

with

(2S,3S)-2-methyl-3-hydroxypentanoyl attached to its cognate RifACP10, RifDH10 also dehydrated this diastereomeric substrate via an syn manner to produce (E)-2-methyl-2-pentenoyl-RifACP10 (Figure 5C) (97). RifDH10 did not dehydrate any of the other three diastereomeric, RifACP10-bound, diketide thioester substrates (97). These findings demonstrated that RifACP10 plays a pivotal role in reversing the stereochemical preference of RifDH10. The detailed mechanism for this ACP-tuned stereospecific dehydration is currently unclear. A recent study on the covalently crosslinked ACP and fatty acid 3-hydroxyacyl-ACP DH obtained by using a mechanism-based crosslinking probe suggested that the DH extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning one of the helices of ACP (98). The subtle difference in protein-protein interaction of ACP and DH could likely lead to opposite binding pattern of the 3-hydroxyacyl substrate to the

enzyme

active

site

and

thereby

reverse

the

stereochemistry

in

dehydration/hydration.

Conclusion and outlook Enzymes have long been renowned for their regio- and stereo-specific catalysis and believed to strictly control the final outcomes of the reactions they catalyze. However, recent studies have demonstrated that the stereochemical outcomes can also be 16


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controlled by the substrates, as illustrated by the examples discussed herein. It is noteworthy that enzymes still play important role in these processes, and this implies diverse evolution strategies for enzymes to produce metabolites of suitable structures. In the biosynthesis of lanthipeptides and sactipeptides, the stereochemistry in the reaction is governed by the intrinsic preference of substrates in the examples discussed. When the final products provide evolutionary advantages (e.g. having antibiotic activity), the enzymes have evolved only to make these reactions more efficiently. If the substrate has no stereochemical preference, evolution selects for the enzymes and/or the substrates to allow certain stereochemistry to be generated, as exemplified in the ketoreduction in hypothemycin biosynthesis (enzyme evolution) and in the dehydration in rifamycin biosynthesis (substrate evolution), respectively. Future studies are expected to unravel the detailed mechanisms how stereochemistry is controlled by substrates in the enzyme active sites, and to reveal more substrate-controlled stereochemical reactions, which might be more general than currently appreciated. These studies will not only expand our understanding of biochemistry but also facilitate bioengineering efforts to produce unnatural metabolites with desired structures and activities.

AUTHOR INFORMATION *

To whom correspondence should be addressed. E-mail: [email protected], phone:

86-21-6564-8172

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ACKNOWLEDGMENTS This work was supported in part by a grant from Fudan University (IDH1615002 to Q.Z). Q.Z. would also like to thank the support of Thousand Talents Program.

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Figure Legends Figure 1. Biosynthesis of lanthipeptides. (A) Lanthionine and methyllanthionine formation, showing the structure of canonical stereochemistry of DL-Lan and DL-MeLan, and the unusual stereochemistry of LL-Lan and LL-MeLan. Xn represents a peptide linker region. (B) Formation of the lacticin 481 ring structure, which was achieved either by modification of the native peptide substrate LctA using the native enzyme LctM (equation i), or by modification of a chimeric peptide (ProcA-LctA, which consists of a ProcA leader and an LctA core peptide) using the highly promiscuous enzyme ProcM (equation ii). (C) Formation of the cytolysin S ring structure by modification of the native peptide substrate CyLs by CylM (equation i), or the three chimeric peptides HalA2-CyLs (equation ii), LtnA2-CyLs (equation iii), and ProcA-CyLs (equation iv), by HalM2, LtnM2, and ProcM, respectively. The unusual LL-MeLan structures are shown in blue. The LL stereochemistry was reversed to the canonical DL stereochemistry by simply introducing a Thr-to-Ala mutation in the T-T-X-X-C motif (equation v), suggesting the second Dhb residue is crucial for the formation of the unusual LL-MeLan. The mutation resulting in an Ala residue in the motif is highlighted by a red circle.

Figure 2. Biosynthesis of sactipeptides. (A) Sactionine formation, showing the structures with two stereochemistries. (B) Structure of sactipeptides, showing the stereochemistries of sactionine residues. The sactionine rings and other rings (disulfide ring, N-to-C amide rings) are shown in black and orange, respectively. 25



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Figure 3. A hypothetic mechanism for the biosynthesis of the two sactionine stereoisomers. The two squares represent the two [4Fe-4S] clusters in sactionine synthetases. RS, radical SAM cluster; A, auxiliary cluster.

Figure 4. Substrate-controlled stereochemistry in ketoreduction catalyzed by HR-PKSs. (A) Biosynthesis of hypothemycin. The precursor DHZ is biosynthesized by the sequential action of a HR-PKS Hpm8 and an NR-PKS Hpm3. (B) Substrate length-controlled stereochemistry of Hpm8 KR, which catalyzes the L-type reduction on diketide substrate (i), and the D-type reduction on substrates longer than diketide (ii). (C) Chemical structure of radicicol and its biosynthetic precursor (R)-monocillin II. (D) L-type reduction on a diketide substrate catalyzed by Rdc5 KR (i) and Hpm8-Rdc5 chimeric KRs (ii), respectively. (E) The chemical structure of epi-DHZ, which is produced by Hpm3 and an engineered Hpm8 containing an Hpm8-Rdc5 chimeric KR.

Figure 5. Substrate-controlled dehydration/hydration catalyzed by RifDH10, which performs catalysis stereospecifically on thioester substrates containing the (2R,3R)-2-methyl-3-hydroxypentanoyl group tethered to N-acetyl cysteamine (NAC) and pantetheine (A), and tethered to EryACP6 (B), and on substrate containing the diastereoisomeric (2S,3S)-2-methyl-3-hydroxypentanoyl group tethered to the native RifACP10 (C). 26


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Substrate-Controlled Stereochemistry in Natural Product Biosynthesis

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