Elucidation of the Cryptic Epimerase Activity of Redox-Inactive Ketoreductase Domains from Modular Polyketide Synthases by Tandem Equilibrium Isotope Exchange
2015-12-17T03:25:09Z (GMT) by
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<sup>0</sup>” domains catalyze the biosynthetically essential epimerization of transient (2<i>R</i>)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2<i>S</i>)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-<sup>2</sup>H]-(2<i>R</i>,3<i>S</i>)-2-methyl-3-hydroxypentanoyl-SACP ([2-<sup>2</sup>H]-<b>3b</b>) with the EryKR3<sup>0</sup> domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP<sup>+</sup> resulted in time- and cofactor-dependent washout of deuterium from <b>3b</b>, as a result of EryKR3<sup>0</sup>-catalyzed epimerization of transiently generated [2-<sup>2</sup>H]-2-methyl-3-ketopentanoyl-ACP (<b>4</b>). Similar results were obtained with redox-inactive PicKR3<sup>0</sup> 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<sup>0</sup> mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR<sup>0</sup> 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.