Directed Mutagenesis Alters the Stereochemistry of Catalysis by Isolated Ketoreductase Domains from the Erythromycin Polyketide Synthase

Abstract

The ketoreductase (KR) domains eryKR1 and eryKR2 from the erythromycin-producing polyketide synthase (PKS) reduce 3-ketoacyl-thioester intermediates with opposite stereospecificity. Modeling of eryKR1 and eryKR2 showed that conserved amino acids previously correlated with production of alternative alcohol configurations lie in the active site. eryKR1 domains mutated at these positions showed an altered stereochemical outcome in reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester. The wild-type eryKR1 domain exclusively gave the (2S, 3R)-3-hydroxy-2-methylpentanoic acid N-acetylcysteamine thioester, while the double mutant (F141W, P144G) gave only the (2S, 3S) isomer, a switch of the alcohol stereochemistry. Mutation of the eryKR2 domain, in contrast, greatly increased the proportion of the wild-type (2R, 3S)-alcohol product. These data confirm the role of key residues in stereocontrol and suggest an additional way to make rational alterations in polyketide antibiotic structure.