O'Hare Helen M, Baerga-Ortiz Abel, Popovic Bojana, Spencer Jonathan B, Leadlay Peter F
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
Chem Biol. 2006 Mar;13(3):287-96. doi: 10.1016/j.chembiol.2006.01.003.
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.
酮还原酶（KR）活性有助于确定模块化聚酮合酶（PKS）产物的立体化学结构。例如，在产生红霉素的PKS的第一个和第二个延伸模块中分别含有的eryKR(1)和eryKR(2)结构域，以相反的立体特异性还原3-酮酰基硫酯中间体。在酮还原酶中已鉴定出与立体化学结果相关的氨基酸基序。我们对eryKR(1)和eryKR(2)中的这些基序进行了饱和诱变，并基于微孔板对这些突变体针对(9R, S)-反式-1-十氢萘酮的活性进行筛选，以鉴定立体控制可能发生改变的候选酶。用(2R, S)-2-甲基-3-氧代戊酸N-乙酰半胱氨酸硫酯对活性突变体进行重新测定，并通过手性高效液相色谱法分析醇产物。发现变体酶对(2R)或(2S)底物的底物选择性发生改变，或者还原的立体特异性发生改变，或两者皆有，这进一步突出了这些基序在立体化学控制中的重要性。
