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H-bonding interactions of Pim TE-catalyzed pimaricin chain release based on MD simulation. (A) Comparison of prereaction conformation of Pim TE-5a and Pim TE-5c. Unnatural substrate 5c is twisted in the substrate channel when cyclized by Pim TE. Substrate 5a is shown in green and substrate 5c in blue. (B) H-bond interactions between Pim TE and substrate 5a. The residues are colored as follows: from the loop I region (blue), from αII and αL2 (gold), and near active sites (red). (C) Key H-bond interactions between C12-COOH and Pim TE in MD simulation of Pim TE-5a. The related residues are highlighted in blue. (D) H-bond interactions between Pim TE and substrate 5c. The residues are colored as follows: from the loop I region (blue), from αII and αL2 (gold), and near active sites (red).
Source publication
Polyketides serve as rich source of therapeutically relevant drug leads. The manipulation of polyketide synthases (PKSs) for generating derivatives with improved activities usually results in substantially reduced yields. Growing evidence suggests that type I PKS thioesterase (TE) domains are key bottlenecks in the biosynthesis of polyene antibioti...
Contexts in source publication
Context 1
... LC-MS/MS and NMR spectroscopy analysis, compound 5d was characterized as 2-hydro-3-hydroxy-4,5-desepoxy-12-decarboxy-12-methyl pimaricin (C 27 H 42 O 9 ) (see the Supporting Information) and considered to be a fully elongated linear decarboxylated pimaricin intermediate. The same TE L170R mutation was also introduced into the wild-type strain S. chattanoogensis L10 (ZYC01, Figures S3 and S5) and accumulated another full-length linear polyketide 5a (m/z = 521.2392 ([M − H] − ), Figure S5D), which was speculated to be the natural intermediate that tethered to the ACP12 domain during the biosynthesis of pimaricin ( Figure 1A). ...
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... same TE L170R mutation was also introduced into the wild-type strain S. chattanoogensis L10 (ZYC01, Figures S3 and S5) and accumulated another full-length linear polyketide 5a (m/z = 521.2392 ([M − H] − ), Figure S5D), which was speculated to be the natural intermediate that tethered to the ACP12 domain during the biosynthesis of pimaricin ( Figure 1A). ...
Context 3
... enzyme models, including S138-covalent connections with both natural C12-COOH 28 and unnatural C12-CH 3 substrates (5c, Δ 2,3 -4,5-desepoxy-12-decarboxy-12-methyl pimaricin), were used for comparative studies of MD simulations (see Figures 1 and S14). These two covalent-binding complexes (Pim TE-5a and Pim TE-5c) were subjected to three times 100 ns MD simulations ( Figures S15 and S16). Two distances were employed to evaluate the productive and unproductive conformations, including the Nε-O25 distance between the nucleophilic oxygen of C25-OH and the Nε nitrogen of H261 in the catalytic triad and the C1-O25 distance between the nucleophilic oxygen of C25-OH and C1 from the substrates. ...
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... further identify the molecular basis of the catalytic bottleneck of Pim TE toward the unnatural C12-CH 3 substrate, the H-bonding and hydrophobic interactions between Pim TE and substrates 5a or 5c were counted ( Figure 5A). The H-bonding interactions near the C12 elbow were analyzed first. ...
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... H-bonding interactions near the C12 elbow were analyzed first. Substrate 5a was observed to be not only H-bonded with Q29, M210, and N214 from αII and αL2 but also with a series of hydrophilic residues (D179, N175, H172, etc.) in the loop I region on the opposite side of the channel ( Figure 5B). Specifically, the C12-COOH carboxylate group in substrate 5a exhibited H-bonding interactions with Q29, Q174, N175, G211, and N214 ( Figure 5C), which balances the hydrophilic network on both sides of the substrate channel. ...
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... 5a was observed to be not only H-bonded with Q29, M210, and N214 from αII and αL2 but also with a series of hydrophilic residues (D179, N175, H172, etc.) in the loop I region on the opposite side of the channel ( Figure 5B). Specifically, the C12-COOH carboxylate group in substrate 5a exhibited H-bonding interactions with Q29, Q174, N175, G211, and N214 ( Figure 5C), which balances the hydrophilic network on both sides of the substrate channel. In contrast, the H-bonding interactions between the unnatural substrate 5c and Pim TE decreased; in addition to an intensive hydrophilic interaction with Asp179 in the loop I region, substrate 5c forms a weak interaction with N214 in αL2 ( Figure 5D). ...
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... the C12-COOH carboxylate group in substrate 5a exhibited H-bonding interactions with Q29, Q174, N175, G211, and N214 ( Figure 5C), which balances the hydrophilic network on both sides of the substrate channel. In contrast, the H-bonding interactions between the unnatural substrate 5c and Pim TE decreased; in addition to an intensive hydrophilic interaction with Asp179 in the loop I region, substrate 5c forms a weak interaction with N214 in αL2 ( Figure 5D). Moreover, in the MD simulation of Pim TE-5a, the hydrophobic cleft that consists of I25, G182, L183, M210, and F262 is well aligned with the C16-C23 polyene moiety ( Figure S19A), facilitating the stable and repeated emergence of the macrolactonization-favorable conformation of substrate 5a. ...
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... nonpolar C12-CH 3 group, instead of the polar carboxyl group, in unnatural substrate 5c could result in unbalanced H-bonding interactions at the C12 elbow and curled substrate 5c docking in Pim TE. Thus, the terminal hydroxyl C25-OH of 5c is proximal to the hydrophilic residues near the active center ( Figure S25), which disturbs the deprotonation of C25-OH by H261 to form a macrocyclized product. Briefly, the TE bottlenecks on the Pim TE-catalyzed chain released an unnatural substrate with the C12-CH 3 cloud can be summarized as follows: (1) holding the appropriate and stable pose for the distal C25-OH from substrate 5c to the active site in a macrolactonization-suitable conformation is difficult because of the lack of accessible H-bond donor residues in αII and αL2 to balance the hydrophilic network at the C12 elbow; (2) the hydrophobic interaction between the polyene moiety and hydrophobic cleft was attenuated upon the macrocyclization of unnatural substrates with C12-CH 3 ; and (3) the distal C25-OH is easily trapped by competitive hydrophilic residues near other active sites rather than at the H261 site to form a macrolactonization-favorable conformation. ...
Citations
... The basis for cyclization vs. hydrolysis by mPKS TEs (Fig. 1A) is poorly understood 6,12 despite published structures of three macrolactone-forming TE domains, including the DEBSIII TE (erythromycin, 14-membered macrolactone 6-deoxyerythronolide B (6-deB)) 13,14 , PikAIV TE (pikromycin, 12-membered 10-deoxymethynolide (10-dml) or 14-membered narbonolide) 15,16 , and PimS4 TE (pimaricin, 26-membered polyene ring) 17 ; one hydrolyzing TE (TmcB TE, linear product leading to tautomycetin) 18 ; and one decarboxylating TE (CurM TE, linear product curacin A) 19 . Biochemical study of DEBSIII TE and PikAIV TE revealed some substrate selectivity in macrolactone formation 8,9,15,[20][21][22][23] . ...
... The exit end of the active site tunnel forms a spacious acyl cavity large enough to accommodate the polyketide substrate as well as the macrolactone or linear carboxylic acid product. The shape and extent of the acyl pocket (helices α2, α6, α7) is the most variable region in TE structures 13,15,17 , and includes several positions where sequences vary (Fig. S1A). No significant structural differences exist between mPKS TE free enzymes and their corresponding (which was not certified by peer review) is the author/funder. ...
... ; https://doi.org/10.1101/2024.06.20.599880 doi: bioRxiv preprint substrate, product or analog complexes 15,17,29 . Thus, substrate selectivity and catalytic outcome -macrolactone formation vs. hydrolysis -are not driven by conformational changes to the active site tunnel or acyl cavity but rather by other TE features such as the shape and chemical environment of the acyl cavity, the substrate structure, and the length, position and amphipathic profile of the lid region 6,13,15,17 15,29 . ...
Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TEDAP) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TEDAP, the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 A from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.
... In deleting modules from amphotericin PKS proteins, care was taken to use junctions that were considered optimal. The likely problem was the TE domain, which has been considered a bottleneck in other engineered PKSs [18]. ...
... Polyene chain-terminating TEs have been studied intensively [18,19]. These domains belong to a distinct phylogenetic group. ...
... They are closely related to each other but not to the erythromycin and pikromycin PKS TE domains. The structure of the pimaricin TE domain has been determined [18]. This indicates that polyene PKS TE domains are adapted to act on their native substrates and may have a lower tolerance towards altered polyketide chains. ...
Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved antibiotics. Here, Streptomyces nodosus was genetically re-programmed to synthesise pentaene analogues of the heptaene amphotericin B. These pentaenes are of interest as surrogate substrates for enzymes catalysing unusual, late-stage biosynthetic modifications. The previous deletion of amphotericin polyketide synthase modules 5 and 6 generated S. nodosus M57, which produces an inactive pentaene. Here, the chain-terminating thioesterase was fused to module 16 to generate strain M57-16TE, in which cycles 5, 6, 17 and 18 are eliminated from the biosynthetic pathway. Another variant of M57 was obtained by replacing modules 15, 16 and 17 with a single 15–17 hybrid module. This gave strain M57-1517, in which cycles 5, 6, 15 and 16 are deleted. M57-16TE and M57-1517 gave reduced pentaene yields. Only M57-1517 delivered its predicted full-length pentaene macrolactone in low amounts. For both mutants, the major pentaenes were intermediates released from modules 10, 11 and 12. Longer pentaene chains were unstable. The novel pentaenes were not glycosylated and were not active against Candida albicans. However, random mutagenesis and screening may yet deliver new antifungal producers from the M57-16TE and M57-1517 strains.
... Modular type I PKS is recognized as one of the mostly studied biosynthetic systems to construct diverse natural products. 36 The BGC olm had 44 contiguous open reading frames (ORFs) within a DNA region of 127.93 kbp (Table S3). It consisted of seven core modular PKS genes (olmA1-olmA7) carrying 17 modules, including a loading module, which catalyzed a series of stepwise condensations of diverse acyl-CoA precursors. ...
... A highly-reducing PKS has KR, DH, and ER domains whereas a partially-reducing PKS lacks either β-keto reductase (KR), dehydratase (DH), or enoyl reductase (ER) domain. The functions of individual domains have been dealt with in detail in other articles [23][24][25][28][29][30][31] and will not be elaborated upon in the current article. In this review, I mostly focus on non-reducing PKSs and the compounds they produce as most of the lichen metabolites connected to their genes are NRPKS-derived. ...
Lichen secondary metabolites have tremendous pharmaceutical and industrial potential. Although more than 1000 metabolites have been reported from lichens, less than 10 have been linked to the genes coding them. The current biosynthetic research focuses strongly on linking molecules to genes as this is fundamental to adapting the molecule for industrial application. Metagenomic-based gene discovery, which bypasses the challenges associated with culturing an organism, is a promising way forward to link secondary metabolites to genes in non-model, difficult-to-culture organisms. This approach is based on the amalgamation of the knowledge of the evolutionary relationships of the biosynthetic genes, the structure of the target molecule, and the biosynthetic machinery required for its synthesis. So far, metagenomic-based gene discovery is the predominant approach by which lichen metabolites have been linked to their genes. Although the structures of most of the lichen secondary metabolites are well-documented, a comprehensive review of the metabolites linked to their genes, strategies implemented to establish this link, and crucial takeaways from these studies is not available. In this review, I address the following knowledge gaps and, additionally, provide critical insights into the results of these studies, elaborating on the direct and serendipitous lessons that we have learned from them.
... Our calculations indicate that substrate tautomerization is crucial in pre-organization of pre-reaction states, as in many biochemical processes. [29][30][31]42,43,52,53 Shape complementarities between the DHP substrate and the protein side chains at NicX's active site are so strict as to allow only linear arrangement of the DHP-O 2 -Fe cluster, supporting conditional 1-electron transfer from pyridine p electron to iron-bound dioxygen. In contrast to the other extradiol cleaving dioxygenases, the iron charge does not change significantly during the dioxygen activation. ...
Characterization of thioesterases (TEs) is an important step in understanding natural product biosynthesis. Studying non-ribosomal peptide synthetase (NRPS) TEs presents a unique set of challenges with specific cloning and expression issues as well as the challenging synthesis of the thioester peptides substrate required for characterization of the TE. In this method, we describe the cloning and expression of NRPS TEs, the synthesis of thioester peptides, and the in vitro biochemical characterization of the enzyme.
Pimaricin is a small polyene macrolide antibiotic and has been broadly used as an antimycotic and antiprotozoal agent in both humans and foods. As a thioesterase in type‐I polyketide synthase, pimTE controls the 26‐m‐r macrolide main chain release in pimaricin biosynthesis. In this work, we sought to determine whether the 6‐m‐r hemiketal formation was linked to pimTE‐catalyzed 26‐m‐r lactonization. Compared to non‐hemiketal TEs, pimTE is characterized by an aspartic acid residue (D179) accessible to the U‐turn motif in the acyl‐enzyme intermediate. Both the covalent docking and molecular dynamics simulations demonstrate that the reactive conformations for macrocyclic lactonization are drastically promoted by the 6‐m‐r hemiketal. Moreover, the small‐model quantum mechanistic calculations suggest that protic residues can significantly accelerate the 6‐m‐r hemiketal cyclization. In addition, the post‐hemiketal molecular dynamic simulations demonstrate that hydrogen‐bonding networks surrounding the substrate U‐turn of the hairpin‐shaped conformation changes significantly when the 6‐m‐r hemiketal is formed. In particular, the R‐hemiketal intermediate is not only catalyzed by the D179 residue, but also twists the hairpin structure to the 26‐m‐r lactonizing pre‐reaction state. By contrast, the S‐hemiketal formation is unlikely catalyzed by D179, which twists the hairpin in an opposite direction. Our results propose that pimTE could be a bi‐functional enzyme, which can synergistically catalyze tandem 6‐m‐r and 26‐m‐r formations during the main‐chain release of pimaricin biosynthesis.
Efficient biosynthesis of microbial bioactive natural products (NPs) is beneficial for the survival of producers, while self‐protection is necessary to avoid self‐harm resulting from over‐accumulation of NPs. The underlying mechanisms for the effective but tolerable production of bioactive NPs is not well understood. Here in the biosynthesis of two fungal polyketide mycotoxins aurovertin E (1) and asteltoxin (4), we show that the cyclases in the gene clusters promote the release of the polyketide backbone, and reveal that a signal peptide is crucial for their subcellular localization and full activity. Meanwhile, the fungus adopts enzymatic acetylation as the major detoxification pathway of 1. If intermediates are over‐produced, the non‐enzymatic shunt pathways work as salvage pathways to avoid excessive accumulation of the toxic metabolites for self‐protection. These findings provided new insights into the interplay of efficient backbone release and multiple detoxification strategies for the production of fungal bioactive NPs.
Efficient biosynthesis of microbial bioactive natural products (NPs) is beneficial for the survival of producers, while self‐protection is necessary to avoid self‐harm resulting from over‐accumulation of NPs. The underlying mechanisms for the effective but tolerable production of bioactive NPs are not well understood. Herein, in the biosynthesis of two fungal polyketide mycotoxins aurovertin E (1) and asteltoxin, we show that the cyclases in the gene clusters promote the release of the polyketide backbone, and reveal that a signal peptide is crucial for their subcellular localization and full activity. Meanwhile, the fungus adopts enzymatic acetylation as the major detoxification pathway of 1. If intermediates are over‐produced, the non‐enzymatic shunt pathways work as salvage pathways to avoid excessive accumulation of the toxic metabolites for self‐protection. These findings provided new insight into the interplay of efficient backbone release and multiple detoxification strategies for the production of fungal bioactive NPs.
