Coupled Methyl Group Epimerization and Reduction by Polyketide Synthase Ketoreductase Domains. Ketoreductase-Catalyzed Equilibrium Isotope Exchange

Incubation of [2-<sup>2</sup>H]-(2<i>S</i>,3<i>R</i>)-2-methyl-3-hydroxy­pentanoyl-SACP ([2-<sup>2</sup>H]-<b>1a</b>) with the epimerizing keto­reductase domain EryKR1 in the presence of a catalytic amount NADP<sup>+</sup> (0.05 equiv) resulted in time- and cofactor-dependent washout of deuterium from <b>1a</b>, as a result of equilibrium isotope exchange of transiently generated [2-<sup>2</sup>H]-2-methyl-3-keto­pentanoyl-ACP. Incubations of [2-<sup>2</sup>H]-(2<i>S</i>,3<i>S</i>)-2-methyl-3-hydroxy-pentanoyl-SACP with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-<sup>2</sup>H]-(2<i>R</i>,3<i>S</i>)-2-methyl-3-hydroxy­pentanoyl-SACP and [2-<sup>2</sup>H]-(2<i>R</i>,3<i>R</i>)-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.