Elucidation of the Cryptic Epimerase Activity of Redox-Inactive Ketoreductase Domains from Modular Polyketide Synthases by Tandem Equilibrium Isotope Exchange

摘要

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 epimeriza-tion of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-~2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-~2H]-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 [2-2H]-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 EryKRl~0 mutants retained the intrinsic epimerase activity of the parent EryKRl 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.