High-Throughput Mutagenesis to Evaluate Models of Stereochemical Control in Ketoreductase Domains from the Erythromycin Polyketide Synthase

SummaryKetoreductase (KR) activities help determine the stereochemistry of the products of modular polyketide synthases (PKSs). For example, domains eryKR1 and eryKR2, contained, respectively, in the first and second extension modules of the erythromycin-producing PKS, reduce 3-ketoacyl-thioester intermediates with opposite stereospecificity. Amino acid motifs that correlate with stereochemical outcome have been identified in KRs. We have used saturation mutagenesis of these motifs in eryKR1 and eryKR2, and a microplate-based screen of such mutants for activity against (9R, S)-trans-1-decalone, to identify candidate enzymes potentially altered in stereocontrol. Active mutants were reassayed with (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester, and the alcohol products were analyzed by chiral HPLC. Variant enzymes were found with either altered substrate selectivity for the (2R) or (2S) substrate or altered stereospecificity of reduction, or both, further highlighting the importance of these motifs in stereochemical control.