title: High-throughput mutagenesis to evaluate models of stereochemical control in ketoreductase domains from the erythromycin polyketide synthase.
abstract: Ketoreductase (KR) activities help determine the stereochemistry of the products of modular polyketide synthases (PKSs). For example, domains eryKR(1) and eryKR(2), 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 eryKR(1) and eryKR(2), 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.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
authors.forename: Helen M
authors.initials: HM
authors.lastname: O'Hare
authors.forename: Abel
authors.initials: A
authors.lastname: Baerga-Ortiz
authors.forename: Bojana
authors.initials: B
authors.lastname: Popovic
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Spencer
authors.forename: Peter F
authors.initials: PF
authors.lastname: Leadlay
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D048630:Polyketide Synthases; D010088:Oxidoreductases