부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.chembiol.2006.01.003 Byline: Helen M. O'Hare (1)(2), Abel Baerga-Ortiz (1), Bojana Popovic (1), Jonathan B. Spencer (2), Peter F. Leadlay (1) Keywords: CHEMBIO Abstract: Ketoreductase (KR) activities help determine the stereochemistry of the products of modular polyketide synthases (PKSs). For example, domains eryKR.sub.1 and eryKR.sub.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.sub.1 and eryKR.sub.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. Author Affiliation: (1) Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom (2) Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom Article History: Received 26 September 2005; Revised 21 December 2005; Accepted 6 January 2006 Article Note: (miscellaneous) Published online: March 24, 2006