The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Ben Shen - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the KetoSynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase–Polyketide Synthase Interface for Leinamycin Biosynthesis
2016Co-Authors: Yong Huang, Gong-li Tang, Guohui Pan, Chin-yuan Chang, Ben ShenAbstract: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
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cycloheximide and actiphenol production in streptomyces sp yim56141 governed by single biosynthetic machinery featuring an acyltransferase less type i Polyketide Synthase
Organic Letters, 2014Co-Authors: Jeremy R Lohman, Shengxiong Huang, Guangrong Zhao, Lihua Xu, Wensheng Xiang, Ben ShenAbstract:Cycloheximide (1) and actiphenol (2) have been isolated from numerous Streptomyces species. Cloning, sequencing, and characterization of a gene cluster from Streptomyces sp. YIM65141 now establish that 1 and 2 production is governed by single biosynthetic machinery. Biosynthesis of 1 features an acyltransferase-less type I Polyketide Synthase to construct its carbon backbone but may proceed via 2 as a key intermediate, invoking a provocative reduction of a phenol to a cyclohexanone moiety in natural product biosynthesis.
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alternative method for site directed mutagenesis of complex Polyketide Synthase in streptomyces albus ja3453
Acta Biochimica et Biophysica Sinica, 2008Co-Authors: Danfeng Song, Ben Shen, Jane M Coughlin, Xiufen Zhou, Chunhua Zhao, Zixin DengAbstract:Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH, encoding a hybrid Polyketide and non-ribosomal peptide enzyme. Tandem ketoSynthase (KS) domains (KS10-1 and KS10-2) were characterized and they show significant homology with known KSs. Using an alternative method that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replications, site-directed mutagenesis of the catalytic triad amino acid cysteines were carried out in each of the tandem KS domains to test the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS10-2 is essential for OZM biosynthesis but KS10-1 is not indispensable and might serve as a “redundant” domain. These results confirmed the existence of an “extra domain” in complex Polyketide Synthase.
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a phosphopantetheinylating Polyketide Synthase producing a linear polyene to initiate enediyne antitumor antibiotic biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Jian Zhang, Steven G Van Lanen, Wen Liu, Pieter C Dorrestein, Neil L Kelleher, Ben ShenAbstract:The enediynes, unified by their unique molecular architecture and mode of action, represent some of the most potent anticancer drugs ever discovered. The biosynthesis of the enediyne core has been predicted to be initiated by a Polyketide Synthase (PKS) that is distinct from all known PKSs. Characterization of the enediyne PKS involved in C-1027 (SgcE) and neocarzinostatin (NcsE) biosynthesis has now revealed that (i) the PKSs contain a central acyl carrier protein domain and C-terminal phosphopantetheinyl transferase domain; (ii) the PKSs are functional in heterologous hosts, and coexpression with an enediyne thioesterase gene produces the first isolable compound, 1,3,5,7,9,11,13-pentadecaheptaene, in enediyne core biosynthesis; and (iii) the findings for SgcE and NcsE are likely shared among all nine-membered enediynes, thereby supporting a common mechanism to initiate enediyne biosynthesis.
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leinamycin biosynthesis revealing unprecedented architectural complexity for a hybrid Polyketide Synthase and nonribosomal peptide synthetase
Chemistry & Biology, 2004Co-Authors: Gong-li Tang, Yi-qiang Cheng, Ben ShenAbstract:A 135,638 bp DNA region that encompasses the leinamycin (LNM) biosynthetic gene cluster was sequenced from Streptomyces atroolivaceus S-140. The boundaries of the lnm cluster were defined by systematic inactivation of open reading frames within the sequenced region. The lnm cluster spans 61.3 kb of DNA and consists of 27 genes encoding nonribosomal peptide synthetase (NRPS), Polyketide Synthase (PKS), hybrid NRPS-PKS, resistance, regulatory, and tailoring enzymes, as well as proteins of unknown function. A model for LNM biosynthesis is proposed, central to which is the LNM hybrid NRPS-PKS megasynthetase consisting of discrete (LnmQ and LnmP) and modular (LnmI) NRPS, acyltransferase-less PKS (LnmG, LnmI, and LnmJ), and PKS modules with unusual domain organization. These studies unveil an unprecedented architectural complexity for the LNM hybrid NRPS-PKS megasynthetase and set the stage to investigate the molecular basis for LNM biosynthesis.
Thomas J Simpson - One of the best experts on this subject based on the ideXlab platform.
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dissecting the component reactions catalyzed by the actinorhodin minimal Polyketide Synthase
Biochemistry, 2007Co-Authors: Pedro Beltranalvarez, John Crosby, Russell J Cox, Thomas J SimpsonAbstract:The actinorhodin (act) minimal Polyketide Synthase (PKS) from Streptomyces coelicolor consists of three proteins: an acyl carrier protein (ACP) and two β-ketoacyl ACP Synthase components known as KSα and KSβ. The act minimal PKS catalyzes at least 18 separate reactions which can be divided into loading, initiation, extension, and cyclization and release phases. Two quantitative kinetic assays were developed and used to measure individual rate and Michaelis constants for loading, initiation and extension steps. In the minimal PKS, the reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-limiting step (kcat = 0.49 min-1, KM = 207 μM). This reaction increases 5-fold in rate in the presence of KSαKSβ (kcat = 2.3 min-1, KM = 215 μM). In the presence of S. coelicolor malonyl CoA:ACP transacylase (MCAT), the rate of loading increases and the kinetic parameters of malonyl-ACP as a substrate of KSαKSβ can be measured (kcat = 20.6 min-1, KM = 2.4 μM). Under these conditions, it appears th...
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solution structure and dynamics of oxytetracycline Polyketide Synthase acyl carrier protein from streptomyces rimosus
Biochemistry, 2003Co-Authors: Stuart C Findlow, John Crosby, Thomas J Simpson, Claire Winsor, Matthew P. CrumpAbstract:Type II Polyketide Synthases (PKSs) utilize a dedicated and essential acyl carrier protein (ACP) in the biosynthesis of a specific Polyketide product. As part of our ongoing studies into the mechanisms and control of Polyketide biosynthesis, we report the second structure of a Polyketide Synthase ACP. In this work, multidimensional, heteronuclear NMR was employed to investigate the structure and dynamics of the ACP involved in the biosynthesis of the commonly prescribed Polyketide antibiotic, oxytetracycline (otc). An ensemble of 28 structures of the 95 amino acid otc ACP (9916Da) was computed by simulated annealing with the inclusion of 1132 experimental restraints. Atomic RMSDs about the mean structure for all 28 models is 0.66 A for backbone atoms, 1.15 A for all heavy atoms (both values calculated for the folded part of the protein (residues 3−80)), and 0.41 A for backbone atoms within secondary structure. Otc ACP adopts the typical right-handed, four-helix fold of currently known ACPs but with the ad...
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ketoSynthase domain probes identify two subclasses of fungal Polyketide Synthase genes
Fungal Genetics and Biology, 1999Co-Authors: Lewis E H Bingle, Thomas J Simpson, Colin M LazarusAbstract:Analysis of fungal Polyketide Synthase gene sequences suggested that these might be divided into two subclasses, designated WA-type and MSAS-type. Two pairs of degenerate PCR primers (LC1 and LC2c, LC3 and LC5c) were designed for the amplification of ketoSynthase domain fragments from fungal PKS genes in each of these subclasses. Both primer pairs were shown to amplify one or more PCR products from the genomes of a range of ascomycetous Deuteromycetes and Southern blot analysis confirmed that the products obtained with each pair of primers emanated from distinct genomic loci. PCR products obtained from Penicillium patulum and Aspergillus parasiticus with the LC1/2c primer pair and from Phoma sp. C2932 with both primer pairs were cloned and sequenced; the deduced protein sequences were highly homologous to the ketoSynthase domains of other fungal PKS genes. Genes from which LC1/2c fragments were amplified (WA-type) were shown by a phylogenetic analysis to be closely related to fungal PKS genes involved in pigment and aflatoxin biosynthetic pathways, whereas the gene from which the LC3/5c fragment was amplified (MSAS-type) was shown to be closely related to genes encoding 6-methylsalicylic acid Synthase (MSAS). The phylogenetic tree strongly supported the division of fungal PKS genes into two subclasses. The LC-series primers may be useful molecular tools to facilitate the cloning of novel fungal Polyketide Synthase genes.
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solution structure of the actinorhodin Polyketide Synthase acyl carrier protein from streptomyces coelicolor a3 2
Biochemistry, 1997Co-Authors: Matthew P. Crump, David A Hopwood, John Crosby, Christopher E Dempsey, John Parkinson, Martin Murray, Thomas J SimpsonAbstract:The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the Polyketide Synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first Polyketide Synthase component for which detailed structural information has been obtained. Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints. The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2. The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3. The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86). The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid Synthase (FAS) ACP, though some structural differences exist. First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E. coli FAS ACP. Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E. coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35). Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing Polyketide chain. The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field.
Matthew P. Crump - One of the best experts on this subject based on the ideXlab platform.
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stabilization and enhanced reactivity of actinorhodin Polyketide Synthase minimal complex in polymer nucleotide coacervate droplets
Chemical Communications, 2012Co-Authors: John Crosby, Tom Treadwell, Michelle Hammerton, Konstantinos Vasilakis, Matthew P. Crump, David S. Williams, Stephen MannAbstract:Compartmentalization of the minimal complex of actinorhodin Polyketide Synthase in coacervate liquid droplets produces enhanced yields of shunt Polyketides under conditions of low and high ionic strength.
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solution structure and dynamics of oxytetracycline Polyketide Synthase acyl carrier protein from streptomyces rimosus
Biochemistry, 2003Co-Authors: Stuart C Findlow, John Crosby, Thomas J Simpson, Claire Winsor, Matthew P. CrumpAbstract:Type II Polyketide Synthases (PKSs) utilize a dedicated and essential acyl carrier protein (ACP) in the biosynthesis of a specific Polyketide product. As part of our ongoing studies into the mechanisms and control of Polyketide biosynthesis, we report the second structure of a Polyketide Synthase ACP. In this work, multidimensional, heteronuclear NMR was employed to investigate the structure and dynamics of the ACP involved in the biosynthesis of the commonly prescribed Polyketide antibiotic, oxytetracycline (otc). An ensemble of 28 structures of the 95 amino acid otc ACP (9916Da) was computed by simulated annealing with the inclusion of 1132 experimental restraints. Atomic RMSDs about the mean structure for all 28 models is 0.66 A for backbone atoms, 1.15 A for all heavy atoms (both values calculated for the folded part of the protein (residues 3−80)), and 0.41 A for backbone atoms within secondary structure. Otc ACP adopts the typical right-handed, four-helix fold of currently known ACPs but with the ad...
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solution structure of the actinorhodin Polyketide Synthase acyl carrier protein from streptomyces coelicolor a3 2
Biochemistry, 1997Co-Authors: Matthew P. Crump, David A Hopwood, John Crosby, Christopher E Dempsey, John Parkinson, Martin Murray, Thomas J SimpsonAbstract:The solution structure of the actinorhodin acyl carrier protein (act apo-ACP) from the Polyketide Synthase (PKS) of Streptomyces coelicolor A3(2) has been determined using 1H NMR spectroscopy, representing the first Polyketide Synthase component for which detailed structural information has been obtained. Twenty-four structures were generated by simulated annealing, employing 699 distance restraints and 94 dihedral angle restraints. The structure is composed, principally, of three major helices (1, 2, and 4), a shorter helix (3) and a large loop region separating helices 1 and 2. The structure is well-defined, except for a portion of the loop region (residues 18-29), the N-terminus (1-4), and a short stretch (57-61) in the loop connecting helices 2 and 3. The RMS distribution of the 24 structures about the average structure is 1.47 A for backbone atoms, 1.84 A for all heavy atoms (residues 5-86), and 1.01 A for backbone atoms over the helical regions (5-18, 41-86). The tertiary fold of act apo-ACP shows a strong structural homology with Escherichia coli fatty acid Synthase (FAS) ACP, though some structural differences exist. First, there is no evidence that act apo-ACP is conformationally averaged between two or more states as observed in E. coli FAS ACP. Second, act apo-ACP shows a disordered N-terminus (residues 1-4) and a longer flexible loop (19-41 with 19-29 disordered) as opposed to E. coli FAS ACP where the N-terminal helix starts at residue 3 and the loop region is three amino acids shorter (16-35). Most importantly, however, although the act apo-ACP structure contains a hydrophobic core, there are in addition a number of buried hydrophilic groups, principally Arg72 and Asn79, both of which are 100% conserved in the PKS ACPs and not the FAS ACPs and may therefore play a role in stabilizing the growing Polyketide chain. The structure-function relationship of act ACP is discussed in the light of these structural data and recent genetic advances in the field.
Sueharu Horinouchi - One of the best experts on this subject based on the ideXlab platform.
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4 hydroxy 3 methyl 6 1 methyl 2 oxoalkyl pyran 2 one synthesis by a type iii Polyketide Synthase from rhodospirillum centenum
ChemBioChem, 2013Co-Authors: Takayoshi Awakawa, Sueharu Horinouchi, Yoshinori Sugai, Kanae Otsutomo, Shukun Ren, Shinji Masuda, Yohei Katsuyama, Yasuo OhnishiAbstract:The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III Polyketide Synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl-CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C(10-14) fatty acyl-CoA unit, one malonyl-CoA unit and two methylmalonyl-CoA units. We identified these products as 4-hydroxy-3-methyl-6-(1-methyl-2-oxoalkyl)pyran-2-ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl-CoA as the extender substrate. In addition, in vitro reactions with (13)C-labeled malonyl-CoA revealed that RpsA produced tetraketide 6-alkyl-4-hydroxy-1,5-dimethyl-2-oxocyclohexa-3,5-diene-1-carboxylic acids from C(14-20) fatty acyl-CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III Polyketide Synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III Polyketide Synthase family.
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alteration of reaction and substrate specificity of a bacterial type iii Polyketide Synthase by site directed mutagenesis
Biochemical Journal, 2002Co-Authors: Nobutaka Funa, Yutaka Ebizuka, Yasuo Ohnishi, Sueharu HorinouchiAbstract:RppA, which belongs to the type III Polyketide Synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III Polyketide Synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone Synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing Polyketide chain. Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.
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properties and substrate specificity of rppa a chalcone Synthase related Polyketide Synthase in streptomyces griseus
Journal of Biological Chemistry, 2002Co-Authors: Nobutaka Funa, Yutaka Ebizuka, Yasuo Ohnishi, Sueharu HorinouchiAbstract:RppA, a chalcone Synthase-related Polyketide Synthase (type III Polyketide Synthase) in the bacterium Streptomyces griseus, catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA. The K(m) value for malonyl-CoA and the k(cat) value for THN synthesis were determined to be 0.93 +/- 0.1 microm and 0.77 +/- 0.04 min(-1), respectively. RppA accepted aliphatic acyl-CoAs with the carbon lengths from C(4) to C(8) as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield alpha-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2',4',6'-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five molecules of malonyl-CoA, suggesting that the size of the active site cavity of RppA is larger than any other chalcone Synthase-related enzymes found so far in plants and bacteria. RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the starter and methylmalonyl-CoA as an extender. Thus, the broad substrate specificity of RppA yields a wide variety of products.
Chaitan Khosla - One of the best experts on this subject based on the ideXlab platform.
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Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase.
Journal of the American Chemical Society, 2020Co-Authors: Kai P. Yuet, Corey W. Liu, Stephen R. Lynch, James Kuo, Wesley Michaels, Robert B. Lee, Abigail E. Mcshane, Brian L. Zhong, Curt R. Fischer, Chaitan KhoslaAbstract:Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line Polyketide Synthase (PKS) that presumably synthesizes an unknown poly...
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complete reconstitution and deorphanization of the 3 mda nocardiosis associated Polyketide Synthase
Journal of the American Chemical Society, 2020Co-Authors: Kai P. Yuet, Stephen R. Lynch, James Kuo, Wesley Michaels, Robert B. Lee, Abigail E. Mcshane, Brian L. Zhong, Curt R. Fischer, Chun Liu, Chaitan KhoslaAbstract:Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line Polyketide Synthase (PKS) that presumably synthesizes an unknown Polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated Polyketide) aglycone by first fully reconstituting the NOCAP Synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to “deorphanize” a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.
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complete reconstitution and deorphanization of the 3 mda nocap nocardiosis associated Polyketide Synthase
bioRxiv, 2020Co-Authors: Kai P. Yuet, Corey W. Liu, Stephen R. Lynch, James Kuo, Wesley Michaels, Robert B. Lee, Abigail E. Mcshane, Brian L. Zhong, Curt R. Fischer, Chaitan KhoslaAbstract:Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly-line Polyketide Synthase (PKS) that presumably synthesizes an unknown natural product. Here, we report the discovery and structure elucidation of the NOCAP (NOCardiosis-Associated Polyketide) aglycone by first fully reconstituting the NOCAP Synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly-line PKS either in vitro or in E. coli, and of using these approaches to deorphanize a complete assembly-line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.
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the conformational flexibility of the acyltransferase from the disorazole Polyketide Synthase is revealed by an x ray free electron laser using a room temperature sample delivery method for serial crystallography
Biochemistry, 2017Co-Authors: Irimpan I Mathews, Chaitan Khosla, Kim H Allison, Thomas Robbins, Artem Y Lyubimov, Monarin Uervirojnangkoorn, Axel T Brunger, Hasan Demirci, S E Mcphillips, Michael HollenbeckAbstract:The crystal structure of the trans-acyltransferase (AT) from the disorazole Polyketide Synthase (PKS) was determined at room temperature to a resolution of 2.5 A using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for Polyketide Synthase reaction dynamics are discussed.
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coupled methyl group epimerization and reduction by Polyketide Synthase ketoreductase domains ketoreductase catalyzed equilibrium isotope exchange
Journal of the American Chemical Society, 2013Co-Authors: Ashish Garg, Chaitan Khosla, David E CaneAbstract:Incubation of [2-2H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP+ (0.05 equiv) resulted in time- and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-2H]-2-methyl-3-ketopentanoyl-ACP. Incubations of [2-2H]-(2S,3S)-2-methyl-3-hydroxy-pentanoyl-SACP with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP and [2-2H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific Polyketide Synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of Polyketide stereocomplexity.