J. Am. Chem. Soc. 136, 10190–10193 (2014)

Ketoreductase domains are responsible for reducing carbonyl groups during polyketide biosynthesis, thus determining the stereochemistry of these centers; some ketoreductases also function as epimerases to invert adjacent methyl-bearing stereocenters. Ketoreductases require NADPH, yet several multimodular polyketide synthases also contain enigmatic 'redox-inactive' homologs of ketoreductases that have lost the capacity to bind NADPH. Garg et al. suspected that these proteins might still perform epimerizations. To test this idea, the authors adapted a previously reported equilibrium isotope exchange assay used to determine whether individual ketoreductases are also epimerases to work as a tandem assay in which an NADPH-dependent, nonepimerizing ketoreductase ('KR' in image) is used to oxidize a representative substrate, and then epimerization of the transiently generated ketoester by the redox-inactive KR0 domain was detected by monitoring washout of deuterium from the reduced substrate. The authors observed that both redox-inactive KR0 domains tested were able to deplete deuterium from the substrate, and tests with a different NADPH-utilizing KR confirmed the generality of the assay. Finally, to explore the epimerization mechanism used by redox-active and redox-inactive ketoreductases, the authors introduced a series of mutations into an epimerase-active KR domain that disrupted NADPH binding, thereby abolishing the native redox activity. These mutants were still able to epimerize the deuterated substrate. These results define a function for this little-studied group of enzymes and rule out an existing NADPH-dependent mechanistic proposal for epimerization.