94 results on '"Macrolides chemistry"'
Search Results
2. Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins.
- Author
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Su L, Hôtel L, Paris C, Chepkirui C, Brachmann AO, Piel J, Jacob C, Aigle B, and Weissman KJ
- Subjects
- Amino Acid Sequence, Macrolides chemistry, Multienzyme Complexes, Substrate Specificity, Synthetic Biology, Macrolides metabolism, Metabolic Engineering, Polyketide Synthases genetics, Polyketide Synthases metabolism
- Abstract
The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites - a reduction in chain length of 14 carbons relative to the 51-membered parental compounds - but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers., (© 2022. The Author(s).)
- Published
- 2022
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3. Synthesis of the C1-C27 Fragment of Stambomycin D Validates Modular Polyketide Synthase-Based Stereochemical Assignments.
- Author
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Lim J, Chintalapudi V, Gudmundsson HG, Tran M, Bernasconi A, Blanco A, Song L, Challis GL, and Anderson EA
- Subjects
- Anti-Bacterial Agents chemistry, Biological Products, Cytochrome P-450 Enzyme System chemistry, Macrolides chemical synthesis, Molecular Structure, Polyketide Synthases chemistry, Streptomyces chemistry, Anti-Bacterial Agents pharmacology, Cytochrome P-450 Enzyme System metabolism, Macrolides chemistry, Polyketide Synthases metabolism, Polyketides chemistry
- Abstract
The stambomycins are a family of bioactive macrolides isolated from Streptomyces ambofaciens . Aside from two stereocenters installed through cytochrome P450 oxidations, their stereochemistry has been predicted by sequence analysis of the polyketide synthase. We report a synthesis of the C1-C27 fragment of stambomycin D, the spectroscopic data of which correlates well with that of the natural product, further validating predictive sequence analysis as a powerful tool for stereochemical assignment of complex polyketide natural products.
- Published
- 2021
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4. Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase.
- Author
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Meng S, Steele AD, Yan W, Pan G, Kalkreuter E, Liu YC, Xu Z, and Shen B
- Subjects
- Animals, Biological Products chemistry, Cysteine metabolism, Cystine chemistry, Cystine metabolism, Humans, Lactams chemical synthesis, Lactams chemistry, Lactams metabolism, Macrolides chemical synthesis, Macrolides chemistry, Macrolides metabolism, Models, Chemical, Molecular Structure, Peptide Synthases metabolism, Streptomyces genetics, Streptomyces metabolism, Substrate Specificity, Sulfides chemistry, Thiazoles chemical synthesis, Thiazoles chemistry, Thiazoles metabolism, Thiones chemical synthesis, Thiones chemistry, Thiones metabolism, src Homology Domains, Biological Products metabolism, Carbon-Sulfur Lyases metabolism, Cysteine analogs & derivatives, Methyltransferases metabolism, Polyketide Synthases metabolism, Sulfides metabolism
- Abstract
Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds., (© 2021. The Author(s).)
- Published
- 2021
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5. Discovery of Polycyclic Macrolide Shuangdaolides by Heterologous Expression of a Cryptic trans -AT PKS Gene Cluster.
- Author
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Liu Y, Zhou H, Shen Q, Dai G, Yan F, Li X, Ren X, Sun Q, Tang YJ, Zhang Y, and Bian X
- Subjects
- Anti-Bacterial Agents chemistry, Macrolides metabolism, Molecular Structure, Multigene Family, Polyketide Synthases metabolism, Protein Synthesis Inhibitors metabolism, Anti-Bacterial Agents biosynthesis, Macrolides chemistry, Polyketide Synthases chemistry, Protein Synthesis Inhibitors chemistry, Streptomyces chemistry
- Abstract
A cryptic trans -acyltransferase polyketide synthase biosynthetic gene cluster sdl (80 kb) from Streptomyces sp. B59 was cloned and transferred into a heterologous host Streptomyces albus J1074, resulting in a class of polycyclic macrolide shuangdaolides A-D ( 1-4 ) and dumulmycin ( 5 ). Heterologous expression and gene inactivation experiments allowed the identification of two biosynthetic intermediates, 6 and 7 , suggesting an unusual multidomain SDR oxidoreductase SdlR in charge of the formation of a rare 2-hydroxycyclopentenone moiety in this class of compounds.
- Published
- 2021
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6. SAXS reveals highly flexible interdomain linkers of tandem acyl carrier protein-thioesterase domains from a fungal nonreducing polyketide synthase.
- Author
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Bunnak W, Winter AJ, Lazarus CM, Crump MP, Race PR, and Wattana-Amorn P
- Subjects
- Circular Dichroism, Macrolides chemistry, Molecular Dynamics Simulation, Protein Domains, Protein Structure, Secondary, Scattering, Small Angle, X-Ray Diffraction, Acyl Carrier Protein chemistry, Fungi enzymology, Polyketide Synthases chemistry, Thiolester Hydrolases chemistry
- Abstract
Menisporopsin A is a fungal bioactive macrocyclic polylactone, the biosynthesis of which requires only reducing (R) and nonreducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions. There is no structural information pertaining to these PKSs. Here, we report the solution characterization of singlet and doublet acyl carrier protein (ACP
2 and ACP1 -ACP2 )-thioesterase (TE) domains from NR-PKS involved in menisporopsin A biosynthesis. Small-angle X-ray scattering (SAXS) studies in combination with homology modelling reveal that these polypeptides adopt a distinctive beads-on-a-string configuration, characterized by the presence of highly flexible interdomain linkers. These models provide a platform for studying domain organization and interdomain interactions in fungal NR-PKSs, which may be of value in directing the design of functionally optimized polyketide scaffolds., (© 2020 Federation of European Biochemical Societies.)- Published
- 2021
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7. Cross-Module Enoylreduction in the Azalomycin F Polyketide Synthase.
- Author
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Zhai G, Wang W, Xu W, Sun G, Hu C, Wu X, Cong Z, Deng L, Shi Y, Leadlay PF, Song H, Hong K, Deng Z, and Sun Y
- Subjects
- Biocatalysis, Macrolides chemistry, Molecular Conformation, Oxidation-Reduction, Polyketide Synthases chemistry, Macrolides metabolism, Polyketide Synthases metabolism
- Abstract
The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis., (© 2020 Wiley-VCH GmbH.)
- Published
- 2020
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8. The biosynthetic pathway to tetromadurin (SF2487/A80577), a polyether tetronate antibiotic.
- Author
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Little RF, Samborskyy M, and Leadlay PF
- Subjects
- Actinobacteria enzymology, Actinobacteria metabolism, Actinomadura, Amino Acid Sequence genetics, Anti-Bacterial Agents chemistry, Base Sequence genetics, Biosynthetic Pathways, Cloning, Molecular, Ethers metabolism, Furans metabolism, Multigene Family genetics, Sequence Alignment, Macrolides chemistry, Polyketide Synthases genetics, Polyketides metabolism
- Abstract
The type I polyketide SF2487/A80577 (herein referred to as tetromadurin) is a polyether tetronate ionophore antibiotic produced by the terrestrial Gram-positive bacterium Actinomadura verrucosospora. Tetromadurin is closely related to the polyether tetronates tetronasin (M139603) and tetronomycin, all of which are characterised by containing a tetronate, cyclohexane, tetrahydropyran, and at least one tetrahydrofuran ring. We have sequenced the genome of Actinomadura verrucosospora to identify the biosynthetic gene cluster responsible for tetromadurin biosynthesis (the mad gene cluster). Based on bioinformatic analysis of the 32 genes present within the cluster a plausible biosynthetic pathway for tetromadurin biosynthesis is proposed. Functional confirmation of the mad gene cluster is obtained by performing in-frame deletions in each of the genes mad10 and mad31, which encode putative cyclase enzymes responsible for cyclohexane and tetrahydropyran formation, respectively. Furthermore, the A. verrucosospora Δmad10 mutant produces a novel tetromadurin metabolite that according to mass spectrometry analysis is analogous to the recently characterised partially cyclised tetronasin intermediate lacking its cyclohexane and tetrahydropyran rings. Our results therefore elucidate the biosynthetic machinery of tetromadurin biosynthesis and lend support for a conserved mechanism of cyclohexane and tetrahydropyran biosynthesis across polyether tetronates., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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9. Elucidation of the Tailoring Steps in Julichrome Biosynthesis by Marine Gastropod Mollusk-Associated Streptomyces sampsonii SCSIO 054.
- Author
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Ji X, Dong Y, Ling C, Zhou Z, Li Q, and Ju J
- Subjects
- Animals, Anti-Bacterial Agents chemistry, Gastropoda metabolism, Molecular Structure, Multigene Family, Anti-Bacterial Agents biosynthesis, Gastropoda chemistry, Macrolides chemistry, Polyketide Synthases metabolism, Streptomyces chemistry
- Abstract
The biosynthetic gene cluster governing the production of antibacterial julichromes was identified from marine gastropod mollusk-associated Streptomyces sampsonii SCSIO 054. Post-PKS assembly/tailoring enzymes JuiL, JuiM, JuiI, and JuiN represent key assembly enzymes. JuiL serves as a ketoreductase. JuiM is an acetyltransferase. JuiI carries out an intriguing biaryl coupling of two julichrome Q
6 units (immediate JuiL, JuiM product) to afford julichrome Q6-6 . JuiN carries out tailoring steps on julichrome Q6-6 , transforming Q6-6 into Q3-3 .- Published
- 2020
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10. An in vitro platform for engineering and harnessing modular polyketide synthases.
- Author
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Miyazawa T, Hirsch M, Zhang Z, and Keatinge-Clay AT
- Subjects
- Bacterial Proteins metabolism, Macrolides chemistry, Polyketide Synthases metabolism, Polyketides chemistry, Protein Engineering, Streptomyces chemistry, Streptomyces genetics, Substrate Specificity, Bacterial Proteins chemistry, Bacterial Proteins genetics, Polyketide Synthases chemistry, Polyketide Synthases genetics, Streptomyces enzymology
- Abstract
To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.
- Published
- 2020
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11. On-Line Polyketide Cyclization into Diverse Medium-Sized Lactones by a Specialized Ketosynthase Domain.
- Author
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Sundaram S, Kim HJ, Bauer R, Thongkongkaew T, Heine D, and Hertweck C
- Subjects
- Bacillus amyloliquefaciens genetics, Bacillus amyloliquefaciens metabolism, Burkholderia genetics, Burkholderia metabolism, Cyclization, Lactones chemistry, Macrolides chemistry, Macrolides metabolism, Models, Molecular, Mutagenesis, Site-Directed, Polyketide Synthases chemistry, Polyketide Synthases genetics, Protein Domains, Substrate Specificity, Bacillus amyloliquefaciens enzymology, Burkholderia enzymology, Lactones metabolism, Polyketide Synthases metabolism
- Abstract
Ketosynthase (KS) domains of modular type I polyketide synthases (PKSs) typically catalyze the Claisen condensation of acyl and malonyl units to form linear chains. In stark contrast, the KS of the rhizoxin PKS branching module mediates a Michael addition, which sets the basis for a pharmacophoric δ-lactone moiety. The precise role of the KS was evaluated by site-directed mutagenesis, chemical probes, and biotransformations. Biochemical and kinetic analyses helped to dissect branching and lactonization reactions and unequivocally assign the entire sequence to the KS. Probing the range of accepted substrates with diverse synthetic surrogates in vitro, we found that the KS tolerates defined acyl chain lengths to produce five- to seven-membered lactones. These results show that the KS is multifunctional, as it catalyzes β-branching and lactonization. Information on the increased product portfolio of the unusual, TE-independent on-line cyclization is relevant for synthetic biology approaches., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2018
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12. Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization.
- Author
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Lowell AN, DeMars MD 2nd, Slocum ST, Yu F, Anand K, Chemler JA, Korakavi N, Priessnitz JK, Park SR, Koch AA, Schultz PJ, and Sherman DH
- Subjects
- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Biocatalysis, Dose-Response Relationship, Drug, Gram-Negative Bacteria drug effects, Gram-Positive Bacteria drug effects, Macrolides chemistry, Macrolides pharmacology, Microbial Sensitivity Tests, Molecular Conformation, Polyketide Synthases chemistry, Polyketides chemistry, Polyketides pharmacology, Structure-Activity Relationship, Tylosin biosynthesis, Tylosin chemistry, Tylosin pharmacology, Anti-Bacterial Agents biosynthesis, Macrolides metabolism, Polyketide Synthases metabolism, Polyketides metabolism, Tylosin analogs & derivatives
- Abstract
Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.
- Published
- 2017
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13. An Iterative Module in the Azalomycin F Polyketide Synthase Contains a Switchable Enoylreductase Domain.
- Author
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Xu W, Zhai G, Liu Y, Li Y, Shi Y, Hong K, Hong H, Leadlay PF, Deng Z, and Sun Y
- Subjects
- Macrolides chemistry, Molecular Conformation, Mutation, Oxidoreductases genetics, Polyketide Synthases genetics, Macrolides metabolism, Oxidoreductases chemistry, Oxidoreductases metabolism, Polyketide Synthases chemistry, Polyketide Synthases metabolism
- Abstract
Detailed analysis of the modular Type I polyketide synthase (PKS) involved in the biosynthesis of the marginolactone azalomycin F in mangrove Streptomyces sp. 211726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of a PKS mutant specifically inactivated in the dehydratase domain of extension-module 1 showed that this module catalyzes two successive elongations with different outcomes. Strikingly, the enoylreductase domain of this module can apparently be "toggled" off and on : it functions in only the second of these two cycles. This novel mechanism expands our understanding of PKS assembly-line catalysis and may explain examples of apparent non-colinearity in other modular PKS systems., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
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14. Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering.
- Author
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Koryakina I, Kasey C, McArthur JB, Lowell AN, Chemler JA, Li S, Hansen DA, Sherman DH, and Williams GJ
- Subjects
- Acyltransferases chemistry, Acyltransferases genetics, Erythromycin chemistry, Macrolides chemistry, Macrolides metabolism, Point Mutation, Polyketide Synthases chemistry, Polyketide Synthases genetics, Polyketides chemistry, Protein Domains, Saccharopolyspora genetics, Saccharopolyspora metabolism, Substrate Specificity, Acyltransferases metabolism, Erythromycin metabolism, Mutagenesis, Site-Directed methods, Polyketide Synthases metabolism, Polyketides metabolism, Saccharopolyspora enzymology
- Abstract
Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.
- Published
- 2017
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15. Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase-Polyketide Synthase Interface for Leinamycin Biosynthesis.
- Author
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Huang Y, Tang GL, Pan G, Chang CY, and Shen B
- Subjects
- Acyl Carrier Protein chemistry, Lactams chemistry, Macrolides chemistry, Molecular Conformation, Peptide Synthases chemistry, Polyketide Synthases chemistry, Protein Domains, Thiazoles chemistry, Thiones chemistry, Acyl Carrier Protein metabolism, Lactams metabolism, Macrolides metabolism, Peptide Synthases metabolism, Polyketide Synthases metabolism, Thiazoles metabolism, Thiones metabolism
- Abstract
Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)-acyl carrier protein (ACP)-KS domains at the NRPS-PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS-ACP-KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide-polyketide natural products.
- Published
- 2016
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16. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase.
- Author
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Vance S, Tkachenko O, Thomas B, Bassuni M, Hong H, Nietlispach D, and Broadhurst W
- Subjects
- Acyl Carrier Protein metabolism, Macrolides metabolism, Polyketide Synthases metabolism, Protein Structure, Secondary, Protein Structure, Tertiary, Acyl Carrier Protein chemistry, Macrolides chemistry, Mycobacterium ulcerans, Polyketide Synthases chemistry
- Abstract
Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4'-phosphopantetheine (Ppant) to create the holo form, (15)N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP., (© 2016 The Author(s).)
- Published
- 2016
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17. Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes.
- Author
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Chiu HT, Weng CP, Lin YC, and Chen KH
- Subjects
- Amino Acid Sequence, Biocatalysis, Macrolides chemistry, Molecular Conformation, Molecular Sequence Data, Polyketide Synthases genetics, Sequence Alignment, Macrolides metabolism, Multigene Family genetics, Polyketide Synthases metabolism
- Abstract
Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-l-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.
- Published
- 2016
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18. Parallel Post-Polyketide Synthase Modification Mechanism Involved in FD-891 Biosynthesis in Streptomyces graminofaciens A-8890.
- Author
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Kudo F, Kawamura K, Furuya T, Yamanishi H, Motegi A, Komatsubara A, Numakura M, Miyanaga A, and Eguchi T
- Subjects
- Chromatography, High Pressure Liquid, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Epoxy Compounds chemistry, Hydroxylation, Kinetics, Macrolides chemistry, Methylation, Methyltransferases metabolism, Multigene Family, Mutagenesis, Site-Directed, Polyketide Synthases genetics, Macrolides metabolism, Polyketide Synthases metabolism, Streptomyces enzymology
- Abstract
To isolate a key polyketide biosynthetic intermediate for the 16-membered macrolide FD-891 (1), we inactivated two biosynthetic genes coding for post-polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD-892 (2), which lacks the epoxide moiety at C8-C9, the hydroxy group at C10, and the O-methyl group at O-25 of FD-891, was isolated from the gfsF/gfsG double-knockout mutant. In addition, 25-O-methyl-FD-892 (3) and 25-O-demethyl-FD-891 (4) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8-C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1, respectively. These results suggest that a parallel post-PKS modification mechanism is involved in FD-891 biosynthesis., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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19. The Phormidolide Biosynthetic Gene Cluster: A trans-AT PKS Pathway Encoding a Toxic Macrocyclic Polyketide.
- Author
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Bertin MJ, Vulpanovici A, Monroe EA, Korobeynikov A, Sherman DH, Gerwick L, and Gerwick WH
- Subjects
- Amino Acid Sequence, Conserved Sequence, Cyanobacteria metabolism, Sequence Alignment, Acyltransferases chemistry, Computational Biology, Macrolides chemistry, Multigene Family, Polyketide Synthases chemistry
- Abstract
Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16-membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans-type acyltransferase (trans-AT) ORFs along with KS-AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans-AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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20. Polyketide synthase chimeras reveal key role of ketosynthase domain in chain branching.
- Author
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Sundaram S, Heine D, and Hertweck C
- Subjects
- Acyltransferases chemistry, Burkholderia metabolism, Macrolides chemistry, Macrolides metabolism, Molecular Conformation, Polyketide Synthases chemistry, Polyketides chemistry, Polyketides metabolism, Acyltransferases metabolism, Burkholderia enzymology, Polyketide Synthases metabolism
- Abstract
Biosynthesis of rhizoxin in Burkholderia rhizoxinica affords an unusual polyketide synthase module with ketosynthase and branching domains that install the δ-lactone, conferring antimitotic activity. To investigate their functions in chain branching, we designed chimeric modules with structurally similar domains from a glutarimide-forming module and a dehydratase. Biochemical, kinetic and mutational analyses reveal a structural role of the accessory domains and multifarious catalytic actions of the ketosynthase.
- Published
- 2015
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21. C-S bond cleavage by a polyketide synthase domain.
- Author
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Ma M, Lohman JR, Liu T, and Shen B
- Subjects
- Carbon-Sulfur Lyases chemistry, Computational Biology, Cysteine chemistry, Drug Design, Lactams chemistry, Macrolides chemistry, Multigene Family, Phylogeny, Polyketides chemistry, Protein Engineering methods, Protein Structure, Tertiary, Streptomyces metabolism, Substrate Specificity, Sulfhydryl Compounds, Thiazoles chemistry, Thiones chemistry, Antibiotics, Antineoplastic chemistry, Carbon chemistry, Polyketide Synthases chemistry, Sulfides chemistry
- Abstract
Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM's antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.
- Published
- 2015
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22. Twofold polyketide branching by a stereoselective enzymatic Michael addition.
- Author
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Heine D, Sundaram S, Bretschneider T, and Hertweck C
- Subjects
- Acyl Coenzyme A chemistry, Antibiotics, Antineoplastic chemistry, Macrolides chemistry, Polyketide Synthases chemistry
- Abstract
The versatility of the branching module of the rhizoxin polyketide synthase was tested in an in vitro enzyme assay with a polyketide mimic and branched (di)methylmalonyl-CoA extender units. Comparison of the products with synthetic reference compounds revealed that the module is able to stereoselectively introduce two branches in one step by a Michael addition-lactonisation sequence, thus expanding the scope of previously studied PKS systems.
- Published
- 2015
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23. Macrodiolide formation by the thioesterase of a modular polyketide synthase.
- Author
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Zhou Y, Prediger P, Dias LC, Murphy AC, and Leadlay PF
- Subjects
- Acylation, Anti-Bacterial Agents chemistry, Cyclization, Macrolides chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Thiolester Hydrolases genetics, Anti-Bacterial Agents biosynthesis, Macrolides metabolism, Polyketide Synthases metabolism, Thiolester Hydrolases metabolism
- Abstract
Elaiophylin is an unusual C2 -symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2 -symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)
- Published
- 2015
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24. Substrate controlled divergence in polyketide synthase catalysis.
- Author
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Hansen DA, Koch AA, and Sherman DH
- Subjects
- Macrolides chemistry, Macrolides metabolism, Molecular Structure, Polyketide Synthases genetics, Polyketides chemistry, Substrate Specificity, Polyketide Synthases metabolism, Polyketides metabolism
- Abstract
Biochemical characterization of polyketide synthases (PKSs) has relied on synthetic substrates functionalized as electrophilic esters to acylate the enzyme and initiate the catalytic cycle. In these efforts, N-acetylcysteamine thioesters have typically been employed for in vitro studies of full PKS modules as well as excised domains. However, substrate engineering approaches to control the catalytic cycle of a full PKS module harboring multiple domains remain underexplored. This study examines a series of alternatively activated native hexaketide substrates on the catalytic outcome of PikAIV, the sixth and final module of the pikromycin (Pik) pathway. We demonstrate the ability to control product formation with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring. This outcome was achieved through modifying the type of hexaketide ester employed, demonstrating the utility of substrate engineering in PKS functional studies and biocatalysis.
- Published
- 2015
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25. Comparative characterization of the lactimidomycin and iso-migrastatin biosynthetic machineries revealing unusual features for acyltransferase-less type I polyketide synthases and providing an opportunity to engineer new analogues.
- Author
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Seo JW, Ma M, Kwong T, Ju J, Lim SK, Jiang H, Lohman JR, Yang C, Cleveland J, Zazopoulos E, Farnet CM, and Shen B
- Subjects
- Antibiotics, Antineoplastic chemistry, Antibiotics, Antineoplastic isolation & purification, Antibiotics, Antineoplastic pharmacology, Bacterial Proteins antagonists & inhibitors, Bacterial Proteins chemistry, Bacterial Proteins genetics, Base Sequence, Bioreactors, Cell Line, Tumor, Cell Survival drug effects, Drug Design, Gene Silencing, Humans, Macrolides chemistry, Macrolides isolation & purification, Macrolides metabolism, Macrolides pharmacology, Molecular Sequence Data, Molecular Structure, Mutant Proteins chemistry, Mutant Proteins metabolism, Neoplasms drug therapy, Piperidones chemistry, Piperidones isolation & purification, Piperidones metabolism, Piperidones pharmacology, Polyketide Synthases antagonists & inhibitors, Polyketide Synthases chemistry, Polyketide Synthases genetics, Polyketides chemistry, Polyketides isolation & purification, Polyketides pharmacology, Protein Engineering, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Stereoisomerism, Streptomyces genetics, Structure-Activity Relationship, Antibiotics, Antineoplastic biosynthesis, Bacterial Proteins metabolism, Models, Biological, Multigene Family, Polyketide Synthases metabolism, Polyketides metabolism, Streptomyces enzymology
- Abstract
Lactimidomycin (LTM, 1) and iso-migrastatin (iso-MGS, 2) belong to the glutarimide-containing polyketide family of natural products. We previously cloned and characterized the mgs biosynthetic gene cluster from Streptomyces platensis NRRL 18993. The iso-MGS biosynthetic machinery featured an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes (MgsIJK). We now report cloning and characterization of the ltm biosynthetic gene cluster from Streptomyces amphibiosporus ATCC 53964, which consists of nine genes that encode an AT-less type I PKS (LtmBCDEFGHL) and one tailoring enzyme (LtmK). Inactivation of ltmE or ltmH afforded the mutant strain SB15001 or SB15002, respectively, that abolished the production of 1, as well as the three cometabolites 8,9-dihydro-LTM (14), 8,9-dihydro-8S-hydroxy-LTM (15), and 8,9-dihydro-9R-hydroxy-LTM (13). Inactivation of ltmK yielded the mutant strain SB15003 that abolished the production of 1, 13, and 15 but led to the accumulation of 14. Complementation of the ΔltmK mutation in SB15003 by expressing ltmK in trans restored the production of 1, as well as that of 13 and 15. These results support the model for 1 biosynthesis, featuring an AT-less type I PKS that synthesizes 14 as the nascent polyketide intermediate and a cytochrome P450 desaturase that converts 14 to 1, with 13 and 15 as minor cometabolites. Comparative analysis of the LTM and iso-MGS AT-less type I PKSs revealed several unusual features that deviate from those of the collinear type I PKS model. Exploitation of the tailoring enzymes for 1 and 2 biosynthesis afforded two analogues, 8,9-dihydro-8R-hydroxy-LTM (16) and 8,9-dihydro-8R-methoxy-LTM (17), that provided new insights into the structure-activity relationship of 1 and 2. While 12-membered macrolides, featuring a combination of a hydroxyl group at C-17 and a double bond at C-8 and C-9 as found in 1, exhibit the most potent activity, analogues with a single hydroxyl or methoxy group at C-8 or C-9 retain most of the activity whereas analogues with double substitutions at C-8 and C-9 lose significant activity.
- Published
- 2014
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26. An evolutionary model encompassing substrate specificity and reactivity of type I polyketide synthase thioesterases.
- Author
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Hari TP, Labana P, Boileau M, and Boddy CN
- Subjects
- Bacteria genetics, Evolution, Molecular, Macrolides chemistry, Macrolides metabolism, Phylogeny, Polyketide Synthases genetics, Substrate Specificity, Thiolester Hydrolases genetics, Bacteria enzymology, Polyketide Synthases metabolism, Thiolester Hydrolases metabolism
- Abstract
Bacterial polyketides are a rich source of chemical diversity and pharmaceutical agents. Understanding the biochemical basis for their biosynthesis and the evolutionary driving force leading to this diversity is essential to take advantage of the enzymes as biocatalysts and to access new chemical diversity for drug discovery. Biochemical characterization of the thioesterase (TE) responsible for 6-deoxyerythronolide macrocyclization shows that a small, evolutionarily accessible change to the substrate can increase the chemical diversity of products, including macrodiolide formation. We propose an evolutionary model in which TEs are by nature non-selective for the type of chemistry they catalyze, producing a range of metabolites. As one metabolite becomes essential for improving fitness in a particular environment, the TE evolves to enrich for that corresponding reactivity. This hypothesis is supported by our phylogenetic analysis, showing convergent evolution of macrodiolide-forming TEs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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27. Chemoenzymatic synthesis of spinosyn A.
- Author
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Kim HJ, Choi SH, Jeon BS, Kim N, Pongdee R, Wu Q, and Liu HW
- Subjects
- Biosynthetic Pathways, Insecticides chemistry, Macrolides chemistry, Saccharopolyspora chemistry, Saccharopolyspora metabolism, Insecticides chemical synthesis, Insecticides metabolism, Macrolides chemical synthesis, Macrolides metabolism, Polyketide Synthases metabolism, Saccharopolyspora enzymology
- Abstract
Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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28. Resorcylic acid lactone biosynthesis relies on a stereotolerant macrocyclizing thioesterase.
- Author
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Heberlig GW, Wirz M, Wang M, and Boddy CN
- Subjects
- Biosynthetic Pathways, Fungi enzymology, Fungi metabolism, Kinetics, Lactones chemistry, Molecular Structure, Resorcinols chemistry, Stereoisomerism, Zearalenone analogs & derivatives, Lactones chemical synthesis, Macrolides chemistry, Polyketide Synthases metabolism, Resorcinols chemical synthesis, Zearalenone chemistry
- Abstract
Zearalenone and radicicol are highly related resorcylic acid lactones with the rare property of having opposite stereochemical configurations of the secondary alcohol involved in lactone formation. The ability of the thioesterases from the zearalenone and radicicol biosynthetic pathways to macrocyclize both D and L configured synthetic substrate analogs was biochemically characterized and showed that both enzymes were highly stereotolerant, macrocyclizing both substrates with similar kinetic parameters. This observed stereotolerance is consistent with a proposed evolution of both natural products from a common ancestral resorcylic acid lactone.
- Published
- 2014
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29. Elucidation of the cryptic epimerase activity of redox-inactive ketoreductase domains from modular polyketide synthases by tandem equilibrium isotope exchange.
- Author
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Garg A, Xie X, Keatinge-Clay A, Khosla C, and Cane DE
- Subjects
- Alcohol Oxidoreductases metabolism, Bacterial Proteins metabolism, Binding Sites, Biocatalysis, Deuterium chemistry, Deuterium metabolism, Macrolides metabolism, NADP chemistry, NADP metabolism, Oxidation-Reduction, Polyketide Synthases metabolism, Protein Structure, Tertiary, Racemases and Epimerases metabolism, Stereoisomerism, Substrate Specificity, Alcohol Oxidoreductases chemistry, Bacterial Proteins chemistry, Macrolides chemistry, Polyketide Synthases chemistry, Racemases and Epimerases chemistry
- Abstract
Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such "KR(0)" domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-3b) with the EryKR3(0) domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP(+) resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR3(0)-catalyzed epimerization of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR3(0) from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR1(0) mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR(0) domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.
- Published
- 2014
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30. Vinylogous chain branching catalysed by a dedicated polyketide synthase module.
- Author
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Bretschneider T, Heim JB, Heine D, Winkler R, Busch B, Kusebauch B, Stehle T, Zocher G, and Hertweck C
- Subjects
- Burkholderia chemistry, Burkholderia genetics, Catalysis, Crystallography, X-Ray, Lactones metabolism, Macrolides chemistry, Mutagenesis, Polyketide Synthases genetics, Protein Structure, Tertiary, Burkholderia enzymology, Models, Molecular, Polyketide Synthases metabolism
- Abstract
Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like β-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching-acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity.
- Published
- 2013
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31. Post-PKS tailoring steps of the spiramycin macrolactone ring in Streptomyces ambofaciens.
- Author
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Nguyen HC, Darbon E, Thai R, Pernodet JL, and Lautru S
- Subjects
- Acylation, Anti-Bacterial Agents chemistry, Bacterial Proteins chemistry, Bacterial Proteins genetics, Formaldehyde chemistry, Gene Silencing, Genes, Bacterial, Glucosamine analogs & derivatives, Glucosamine chemistry, Glycosylation, Hexosamines chemistry, Macrolides chemistry, Oxidation-Reduction, Sequence Deletion, Species Specificity, Spiramycin chemistry, Streptomyces genetics, Time Factors, Anti-Bacterial Agents biosynthesis, Polyketide Synthases chemistry, Spiramycin biosynthesis, Streptomyces chemistry
- Abstract
Spiramycins are clinically important 16-member macrolide antibiotics produced by Streptomyces ambofaciens. Biosynthetic studies have established that the earliest lactonic intermediate in spiramycin biosynthesis, the macrolactone platenolide I, is synthesized by a type I modular polyketide synthase (PKS). Platenolide I then undergoes a series of post-PKS tailoring reactions yielding the final products, spiramycins I, II, and III. We recently characterized the post-PKS glycosylation steps of spiramycin biosynthesis in S. ambofaciens. We showed that three glycosyltransferases, Srm5, Srm29, and Srm38, catalyze the successive attachment of the three carbohydrates mycaminose, forosamine, and mycarose, respectively, with the help of two auxiliary proteins, Srm6 and Srm28. However, the enzymes responsible for the other tailoring steps, namely, the C-19 methyl group oxidation, the C-9 keto group reduction, and the C-3 hydroxyl group acylation, as well as the timing of the post-PKS tailoring reactions, remained to be established. In this study, we show that Srm13, a cytochrome P450, catalyzes the oxidation of the C-19 methyl group into a formyl group and that Srm26 catalyzes the reduction of the C-9 keto group, and we propose a timeline for spiramycin-biosynthetic post-PKS tailoring reactions.
- Published
- 2013
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32. Post-polyketide synthase steps in iso-migrastatin biosynthesis, featuring tailoring enzymes with broad substrate specificity.
- Author
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Ma M, Kwong T, Lim SK, Ju J, Lohman JR, and Shen B
- Subjects
- Molecular Structure, Streptomyces enzymology, Streptomyces genetics, Substrate Specificity, Biosynthetic Pathways, Macrolides chemistry, Piperidones chemistry, Polyketide Synthases chemistry
- Abstract
The iso-migrastatin (iso-MGS) biosynthetic gene cluster from Streptomyces platensis NRRL 18993 consists of 11 genes, featuring an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes MgsIJK. Systematic inactivation of mgsIJK in S. platensis enabled us to (i) identify two nascent products of the iso-MGS AT-less type I PKS, establishing an unprecedented novel feature for AT-less type I PKSs, and (ii) account for the formation of all known post-PKS biosynthetic intermediates generated by the three tailoring enzymes MgsIJK, which possessed significant substrate promiscuities.
- Published
- 2013
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33. Investigation of fungal iterative polyketide synthase functions using partially assembled intermediates.
- Author
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Gao Z, Wang J, Norquay AK, Qiao K, Tang Y, and Vederas JC
- Subjects
- Acetylcysteine chemistry, Biocatalysis, Macrolides chemistry, Molecular Structure, Sulfhydryl Compounds chemistry, Acetylcysteine metabolism, Hypocreales enzymology, Macrolides metabolism, Polyketide Synthases metabolism, Sulfhydryl Compounds metabolism
- Abstract
Iterative polyketide synthases (PKSs) are large, multifunctional enzymes that resemble eukaryotic fatty acid synthases but can make highly functionalized secondary metabolites using complex and unresolved programming rules. During biosynthesis of the kinase inhibitor hypothemycin by Hypomyces subiculosus , a highly reducing iterative PKS, Hpm8, cooperates with a nonreducing iterative PKS, Hpm3, to construct the advanced intermediate dehydrozearalenol (DHZ). The identity of putative intermediates in the formation of the highly reduced hexaketide portion of DHZ were confirmed by incorporation of (13)C-labeled N-acetylcysteamine (SNAC) thioesters using the purified enzymes. The results show that Hpm8 can accept SNAC thioesters of intermediates that are ready for transfer from its acyl carrier protein domain to its ketosynthase domain and assemble them into DHZ in cooperation with Hpm3. Addition of certain structurally modified analogues of intermediates to Hpm8 and Hpm3 can produce DHZ derivatives.
- Published
- 2013
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34. 6-Deoxyerythronolide B synthase thioesterase-catalyzed macrocyclization is highly stereoselective.
- Author
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Pinto A, Wang M, Horsman M, and Boddy CN
- Subjects
- Catalysis, Cyclization, Erythromycin chemistry, Molecular Structure, Stereoisomerism, Erythromycin analogs & derivatives, Macrolides chemistry, Polyketide Synthases metabolism
- Abstract
Macrocyclic polyketide natural products are an indispensable source of therapeutic agents. The final stage of their biosynthesis, macrocyclization, is catalyzed regio- and stereoselectively by a thioesterase. A panel of substrates were synthesized to test their specificity for macrocyclization by the erythromycin polyketide synthase TE (DEBS TE) in vitro. It was shown that DEBS TE is highly stereospecific, successfully macrocyclizing a 14-member ring substrate with an R configured O-nucleophile, and highly regioselective, generating exclusively the 14-member lactone over the 12-member lactone.
- Published
- 2012
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35. Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS.
- Author
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Bonnett SA, Rath CM, Shareef AR, Joels JR, Chemler JA, Håkansson K, Reynolds K, and Sherman DH
- Subjects
- Acyl Coenzyme A metabolism, Anti-Bacterial Agents chemistry, Catalytic Domain, Hydrolysis, Kinetics, Macrolides chemistry, Mutation, Polyketide Synthases genetics, Polyketide Synthases metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Substrate Specificity, Acyl Coenzyme A chemistry, Anti-Bacterial Agents biosynthesis, Fourier Analysis, Macrolides metabolism, Mass Spectrometry, Polyketide Synthases chemistry
- Abstract
Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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36. The stereochemistry of complex polyketide biosynthesis by modular polyketide synthases.
- Author
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Kwan DH and Schulz F
- Subjects
- Acyltransferases metabolism, Molecular Structure, Stereoisomerism, Macrolides chemistry, Macrolides metabolism, Polyketide Synthases metabolism
- Abstract
Polyketides are a diverse class of medically important natural products whose biosynthesis is catalysed by polyketide synthases (PKSs), in a fashion highly analogous to fatty acid biosynthesis. In modular PKSs, the polyketide chain is assembled by the successive condensation of activated carboxylic acid-derived units, where chain extension occurs with the intermediates remaining covalently bound to the enzyme, with the growing polyketide tethered to an acyl carrier domain (ACP). Carboxylated acyl-CoA precursors serve as activated donors that are selected by the acyltransferase domain (AT) providing extender units that are added to the growing chain by condensation catalysed by the ketosynthase domain (KS). The action of ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) activities can result in unreduced, partially reduced, or fully reduced centres within the polyketide chain depending on which of these enzymes are present and active. The PKS-catalysed assembly process generates stereochemical diversity, because carbon-carbon double bonds may have either cis- or trans- geometry, and because of the chirality of centres bearing hydroxyl groups (where they are retained) and branching methyl groups (the latter arising from use of propionate extender units). This review shall cover the studies that have determined the stereochemistry in many of the reactions involved in polyketide biosynthesis by modular PKSs.
- Published
- 2011
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37. Biosynthesis: a twist in the tail.
- Author
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Weissman KJ
- Subjects
- Biosynthetic Pathways, Catalysis, Cyclization, Molecular Structure, Polyketide Synthases biosynthesis, Polyketide Synthases genetics, Stereoisomerism, Streptomyces enzymology, Streptomyces genetics, Streptomyces metabolism, Macrolides chemistry, Polyketide Synthases physiology, Pyrans chemistry, Spiro Compounds chemistry
- Published
- 2011
- Full Text
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38. Molecular basis of elansolid biosynthesis: evidence for an unprecedented quinone methide initiated intramolecular Diels-Alder cycloaddition/macrolactonization.
- Author
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Dehn R, Katsuyama Y, Weber A, Gerth K, Jansen R, Steinmetz H, Höfle G, Müller R, and Kirschning A
- Subjects
- Catalysis, Cyclization, Molecular Structure, Multigene Family, Stereoisomerism, Indolequinones chemistry, Macrolides chemistry, Macrolides metabolism, Polyketide Synthases chemistry, Polyketide Synthases metabolism
- Published
- 2011
- Full Text
- View/download PDF
39. Identification of a bioactive 51-membered macrolide complex by activation of a silent polyketide synthase in Streptomyces ambofaciens.
- Author
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Laureti L, Song L, Huang S, Corre C, Leblond P, Challis GL, and Aigle B
- Subjects
- Cell Line, Tumor, Cell Proliferation drug effects, Gene Silencing, Humans, Macrolides metabolism, Multigene Family, Streptomyces enzymology, Transcriptional Activation, Macrolides chemistry, Macrolides pharmacology, Polyketide Synthases genetics, Streptomyces genetics
- Abstract
There is a constant need for new and improved drugs to combat infectious diseases, cancer, and other major life-threatening conditions. The recent development of genomics-guided approaches for novel natural product discovery has stimulated renewed interest in the search for natural product-based drugs. Genome sequence analysis of Streptomyces ambofaciens ATCC23877 has revealed numerous secondary metabolite biosynthetic gene clusters, including a giant type I modular polyketide synthase (PKS) gene cluster, which is composed of 25 genes (nine of which encode PKSs) and spans almost 150 kb, making it one of the largest polyketide biosynthetic gene clusters described to date. The metabolic product(s) of this gene cluster are unknown, and transcriptional analyses showed that it is not expressed under laboratory growth conditions. The constitutive expression of a regulatory gene within the cluster, encoding a protein that is similar to Large ATP binding of the LuxR (LAL) family proteins, triggered the expression of the biosynthetic genes. This led to the identification of four 51-membered glycosylated macrolides, named stambomycins A-D as metabolic products of the gene cluster. The structures of these compounds imply several interesting biosynthetic features, including incorporation of unusual extender units into the polyketide chain and in trans hydroxylation of the growing polyketide chain to provide the hydroxyl group for macrolide formation. Interestingly, the stambomycins possess promising antiproliferative activity against human cancer cell lines. Database searches identify genes encoding LAL regulators within numerous cryptic biosynthetic gene clusters in actinomycete genomes, suggesting that constitutive expression of such pathway-specific activators represents a powerful approach for novel bioactive natural product discovery.
- Published
- 2011
- Full Text
- View/download PDF
40. Enzymatic extender unit generation for in vitro polyketide synthase reactions: structural and functional showcasing of Streptomyces coelicolor MatB.
- Author
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Hughes AJ and Keatinge-Clay A
- Subjects
- Adenosine Monophosphate chemistry, Adenosine Monophosphate metabolism, Amino Acid Motifs, Biocatalysis, Catalytic Domain, Coenzyme A chemistry, Coenzyme A metabolism, Crystallography, X-Ray, Cysteamine chemistry, Cysteamine metabolism, Macrolides chemistry, Macrolides metabolism, Models, Molecular, Pantetheine chemistry, Pantetheine metabolism, Pyrones chemistry, Pyrones metabolism, Substrate Specificity, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Coenzyme A Ligases chemistry, Coenzyme A Ligases metabolism, Polyketide Synthases metabolism, Streptomyces coelicolor enzymology
- Abstract
In vitro experiments with modular polyketide synthases (PKSs) are often limited by the availability of polyketide extender units. To determine the polyketide extender units that can be biocatalytically accessed via promiscuous malonyl-CoA ligases, structural and functional studies were conducted on Streptomyces coelicolor MatB. We demonstrate that this adenylate-forming enzyme is capable of producing most CoA-linked polyketide extender units as well as pantetheine- and N-acetylcysteamine-linked analogs useful for in vitro PKS studies. Two ternary product complex structures, one containing malonyl-CoA and AMP and the other containing (2R)-methylmalonyl-CoA and AMP, were solved to 1.45 Å and 1.43 Å resolution, respectively. MatB crystallized in the thioester-forming conformation, making extensive interactions with the bound extender unit products. This first structural characterization of an adenylate-forming enzyme that activates diacids reveals the molecular details for how malonate and its derivatives are accepted. The orientation of the α-methyl group of bound (2R)-methylmalonyl-CoA, indicates that it is necessary to epimerize α-substituted extender units formed by MatB before they can be accepted by PKS acyltransferase domains. We demonstrate the in vitro incorporation of methylmalonyl groups ligated by MatB to CoA, pantetheine, or N-acetylcysteamine into a triketide pyrone by the terminal module of the 6-deoxyerythronolide B synthase. Additionally, a means for quantitatively monitoring certain in vitro PKS reactions using MatB is presented., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
41. Classification, prediction, and verification of the regioselectivity of fungal polyketide synthase product template domains.
- Author
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Li Y, Image II, Xu W, Image I, Tang Y, and Image I
- Subjects
- Amino Acid Sequence, Macrolides chemistry, Macrolides metabolism, Phylogeny, Polyketide Synthases classification, Protein Structure, Tertiary, Reproducibility of Results, Stereoisomerism, Structure-Activity Relationship, Substrate Specificity, Computational Biology, Fungi enzymology, Polyketide Synthases chemistry, Polyketide Synthases metabolism
- Abstract
The fungal iterative nonreducing polyketide synthases (NRPKSs) synthesize aromatic polyketides, many of which have important biological activities. The product template domains (PT) embedded in the multidomain NRPKSs mediate the regioselective cyclization of the highly reactive polyketide backbones and dictate the final structures of the products. Understanding the sequence-activity relationships of different PT domains is therefore an important step toward the prediction of polyketide structures from NRPKS sequences and can enable the genome mining of hundreds of cryptic NRPKSs uncovered via genome sequencing. In this work, we first performed phylogenetic analysis of PT domains from NRPKSs of known functions and showed that the PT domains can be classified into five groups, with each group corresponding to a unique product size or cyclization regioselectivity. Group V contains the formerly unverified PT domains that were identified as C6-C11 aldol cyclases. The regioselectivity of PTs from this group were verified by product-based assays using the PT domain excised from the asperthecin AptA NRPKS. When combined with dissociated PKS4 minimal PKS, or replaced the endogenous PKS4 C2-C7 PT domain in a hybrid NRPKS, AptA-PT directed the C6-C11 cyclization of the nonaketide backbone to yield a tetracyclic pyranoanthraquinone 4. Extensive NMR analysis verified that the backbone of 4 was indeed cyclized with the expected regioselectivity. The PT phylogenetic analysis was then expanded to include approximately 100 PT sequences from unverified NRPKSs. Using the assays developed for AptA-PT, the regioselectivities of additional PT domains were investigated and matched to those predicted by the phylogenetic classifications.
- Published
- 2010
- Full Text
- View/download PDF
42. Biosynthesis of polyketides by trans-AT polyketide synthases.
- Author
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Piel J
- Subjects
- Biological Products chemistry, Lactones chemical synthesis, Molecular Structure, Stereoisomerism, Biological Products biosynthesis, Macrolides chemistry, Macrolides metabolism, Polyketide Synthases metabolism
- Abstract
This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that have recently been recognized as one of the major group of proteins involved in the production of bioactive polyketides. 436 references are cited.
- Published
- 2010
- Full Text
- View/download PDF
43. Cyclization of aromatic polyketides from bacteria and fungi.
- Author
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Zhou H, Li Y, and Tang Y
- Subjects
- Cyclization, Models, Molecular, Molecular Structure, Protein Conformation, Bacteria chemistry, Fungi chemistry, Macrolides chemistry, Polyketide Synthases metabolism
- Published
- 2010
- Full Text
- View/download PDF
44. Peering into the black box of fungal polyketide biosynthesis.
- Author
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Weissman KJ
- Subjects
- Fungi chemistry, Macrolides chemistry, Models, Molecular, Polyketide Synthases chemistry, Fungi metabolism, Macrolides metabolism, Polyketide Synthases metabolism
- Published
- 2010
- Full Text
- View/download PDF
45. Synthetic chain terminators off-load intermediates from a type I polyketide synthase.
- Author
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Tosin M, Betancor L, Stephens E, Li WM, Spencer JB, and Leadlay PF
- Subjects
- Cysteamine chemistry, Macrolides chemistry, Malonates chemistry, Malonates metabolism, Molecular Structure, Pantetheine chemistry, Recombinant Proteins metabolism, Substrate Specificity, Cysteamine metabolism, Macrolides metabolism, Pantetheine metabolism, Polyketide Synthases metabolism
- Abstract
Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily-analysed form. We prepared a series of nonhydrolysable pantetheine and N-acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off-load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6-deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC-HR-ESI-MS, are the first of their kind and prove that small-molecule chain terminators can be used as convenient probes of the biosynthetic process.
- Published
- 2010
- Full Text
- View/download PDF
46. Genetic engineering of macrolide biosynthesis: past advances, current state, and future prospects.
- Author
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Park SR, Han AR, Ban YH, Yoo YJ, Kim EJ, and Yoon YJ
- Subjects
- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents metabolism, Bacteria genetics, Bacteria metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Drug Design, Genes, Bacterial, Glycosylation, Macrolides chemistry, Metabolic Networks and Pathways genetics, Molecular Structure, Oxidation-Reduction, Polyketide Synthases metabolism, Protein Engineering, Substrate Specificity, Anti-Bacterial Agents biosynthesis, Genetic Engineering, Macrolides metabolism, Polyketide Synthases genetics
- Abstract
Polyketides comprise one of the major families of natural products. They are found in a wide variety of bacteria, fungi, and plants and include a large number of medically important compounds. Polyketides are biosynthesized by polyketide synthases (PKSs). One of the major groups of polyketides are the macrolides, the activities of which are derived from the presence of a macrolactone ring to which one or more 6-deoxysugars are attached. The core macrocyclic ring is biosynthesized from acyl-CoA precursors by PKS. Genetic manipulation of PKS-encoding genes can result in predictable changes in the structure of the macrolactone component, many of which are not easily achieved through standard chemical derivatization or total synthesis. Furthermore, many of the changes, including post-PKS modifications such as glycosylation and oxidation, can be combined for further structural diversification. This review highlights the current state of novel macrolide production with a focus on the genetic engineering of PKS and post-PKS tailoring genes. Such engineering of the metabolic pathways for macrolide biosynthesis provides attractive alternatives for the production of diverse non-natural compounds. Other issues of importance, including the engineering of precursor pathways and heterologous expression of macrolide biosynthetic genes, are also considered.
- Published
- 2010
- Full Text
- View/download PDF
47. Engineered biosynthesis of plant polyketides: structure-based and precursor-directed approach.
- Author
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Abe I
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Plants chemistry, Plants genetics, Polyketide Synthases chemistry, Protein Engineering trends, Sequence Alignment, Substrate Specificity, Macrolides chemistry, Macrolides metabolism, Plants enzymology, Polyketide Synthases genetics, Polyketide Synthases metabolism, Protein Engineering methods
- Abstract
Pentaketide chromone synthase (PCS) and octaketide synthase (OKS) are novel plant-specific type III polyketide synthases (PKSs) obtained from Aloe arborescens. Recombinant PCS expressed in Escherichia coli catalyzes iterative condensations of five molecules of malonyl-CoA to produce a pentaketide 5,7-dihydroxy-2-methylchromone, while recombinant OKS carries out sequential condensations of eight molecules of malonyl-CoA to yield octaketides SEK4 and SEK4b, the longest polyketides produced by the structurally simple type III PKS. The amino acid sequences of PCS and OKS are 91% identical, sharing 50-60% identity with those of other chalcone synthase (CHS) superfamily type III PKSs of plant origin. One of the most characteristic features is that the conserved active-site Thr197 of CHS (numbering in Medicago sativa CHS) is uniquely replaced with Met207 in PCS and with Gly207 in OKS, respectively. Site-directed mutagenesis and X-ray crystallographic analyses demonstrated that the chemically inert single residue lining the active-site cavity controls the polyketide chain length and the product specificity depending on the steric bulk of the side chain. On the basis of the crystal structures, an F80A/Y82A/M207G triple mutant of the pentaketide-producing PCS was constructed and shown to catalyze condensations of nine molecules of malonyl-CoA to produce an unnatural novel nonaketide naphthopyrone, whereas an N222G mutant of the octaketides-producing OKS yielded a decaketide benzophenone SEK15 from ten molecules of malonyl-CoA. On the other hand, the type III PKSs exhibited broad substrate specificities and catalytic potential. OKS accepted p-coumaroyl-CoA as a starter substrate to produce an unnatural novel C19 hexaketide stilbene and a C21 heptaketide chalcone. Remarkably, the C21 chalcone-forming activity was dramatically increased in the structure-guided OKS N222G mutant. In addition, OKS N222G mutant also yielded unnatural novel polyketides from phenylacetyl-CoA and benzoyl-CoA as a starter substrate. These results suggested that the engineered biosynthesis of plant polyketides by combination of the structure-based and the precursor-directed approach would lead to further production of chemically and structurally divergent unnatural novel polyketides.
- Published
- 2010
- Full Text
- View/download PDF
48. [Polyketide antibiotics produced by polyketide synthase in streptomyces--a review].
- Author
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Chen M, Wang G, Dai S, Xie L, and Li X
- Subjects
- Animals, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Bacterial Proteins genetics, Macrolides chemistry, Macrolides pharmacology, Molecular Sequence Data, Polyketide Synthases genetics, Streptomyces chemistry, Streptomyces enzymology, Streptomyces genetics, Anti-Bacterial Agents metabolism, Bacterial Proteins metabolism, Macrolides metabolism, Polyketide Synthases metabolism, Streptomyces metabolism
- Abstract
Polyketides have played an important role in antibiotic drug discovery with most antibacterial drugs being derived from a natural product or natural product lead. Furthermore, the biosynthetic gene clusters for numerous bioactive polyketides have been intensively studied over the past 15 years. This paper focuses on the polyketide drugs approved by US-FDA and takes a general view in the antibiotics produced by polyketide synthase in streptomyces.
- Published
- 2009
49. In vivo and in vitro analysis of the hedamycin polyketide synthase.
- Author
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Das A and Khosla C
- Subjects
- Acyl Carrier Protein metabolism, Antibiotics, Antineoplastic chemistry, Macrolides chemistry, Multigene Family, Polyketide Synthases genetics, Streptomyces enzymology, Streptomyces genetics, Anthraquinones chemistry, Antibiotics, Antineoplastic biosynthesis, Polyketide Synthases metabolism
- Abstract
Hedamycin is an antitumor polyketide antibiotic with unusual biosynthetic features. Earlier sequence analysis of the hedamycin biosynthetic gene cluster implied a role for type I and type II polyketide synthases (PKSs). We demonstrate that the hedamycin minimal PKS can synthesize a dodecaketide backbone. The ketosynthase (KS) subunit of this PKS has specificity for both type I and type II acyl carrier proteins (ACPs) with which it collaborates during chain initiation and chain elongation, respectively. The KS receives a C(6) primer unit from the terminal ACP domain of HedU (a type I PKS protein) directly and subsequently interacts with the ACP domain of HedE (a type II PKS protein) during the process of chain elongation. HedE is a bifunctional protein with both ACP and aromatase activity. Its aromatase domain can modulate the chain length specificity of the minimal PKS. Chain length can also be influenced by HedA, the C-9 ketoreductase. While co-expression of the hedamycin minimal PKS and a chain-initiation module from the R1128 PKS yields an isobutyryl-primed decaketide, the orthologous PKS subunits from the hedamycin gene cluster itself are unable to prime the minimal PKS with a nonacetyl starter unit. Our findings provide new insights into the mechanism of chain initiation and elongation by type II PKSs.
- Published
- 2009
- Full Text
- View/download PDF
50. MapsiDB: an integrated web database for type I polyketide synthases.
- Author
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Tae H, Sohng JK, and Park K
- Subjects
- Database Management Systems, Databases, Genetic, Internet, Macrolides chemistry, Macrolides classification, Macrolides metabolism, Polyketide Synthases genetics, Polyketide Synthases metabolism, User-Computer Interface, Databases, Protein, Polyketide Synthases chemistry
- Abstract
Polyketides have diverse biological activities, including pharmacological functions such as antibiotic, antitumor and agrochemical properties. They are biosynthesized from short carboxylic acid precursors by polyketide synthases (PKSs). As natural polyketide products include many clinically important drugs and the volume of data on polyketides is rapidly increasing, the development of a database system to manage polyketide data is essential. MapsiDB is an integrated web database formulated to contain data on type I polyketides and their PKSs, including domain and module composition and related genome information. Data on polyketides were collected from journals and online resources and processed with analysis programs. Web interfaces were utilized to construct and to access this database, allowing polyketide researchers to add their data to this database and to use it easily.
- Published
- 2009
- Full Text
- View/download PDF
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