title: Stereochemistry of reductions catalyzed by methyl-epimerizing ketoreductase domains of polyketide synthases.
abstract: Ketoreductase (KR) domains from modular polyketide synthases (PKSs) catalyze the reduction of 2-methyl-3-ketoacyl acyl carrier protein (ACP) substrates and in certain cases epimerization of the 2-methyl group as well. The structural and mechanistic basis of epimerization is poorly understood, and only a small number of such KRs been studied. In this work, we studied three recombinant KR domains with putative epimerase activity: NysKR1 from module 1 of the nystatin PKS, whose stereospecificity can be predicted from both the protein sequence and the product structure; RifKR7 from module 7 of the rifamycin PKS, whose stereospecificity cannot be predicted from the protein sequence; and RifKR10 from module 10 of the rifamycin PKS, whose specificity is unclear from both the sequence and the structure. Each KR was individually incubated with NADPH and (2R)- or (2RS)-2-methyl-3-ketopentanoyl-ACP generated enzymatically in situ or via chemoenzymatic synthesis, respectively. Chiral GC-MS analysis revealed that each KR stereospecifically produced the corresponding (2S,3S)-2-methyl-3-hydroxypentanoyl-ACP in which the 2-methyl substituent had undergone KR-catalyzed epimerization. Thus, our results have led to the identification of a prototypical set of KR domains that generate (2S,3S)-2-methyl-3-hydroxyacyl products in the course of polyketide biosynthesis.
authors: Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
authors: Young-Ok
authors: YO
authors: You
authors: Chaitan
authors: C
authors: Khosla
authors: David E
authors: DE
authors: Cane
mesh_terms: D055162:Biocatalysis; D002352:Carrier Proteins / Q000096:biosynthesis; D010084:Oxidation-Reduction; D048630:Polyketide Synthases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism
chemical_list: D002352:Carrier Proteins; D048630:Polyketide Synthases
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