Javidpour Pouya, Bruegger Joel, Srithahan Supawadee, Korman Tyler P, Crump Matthew P, Crosby John, Burkart Michael D, Tsai Shiou-Chuan
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA.
Chem Biol. 2013 Oct 24;20(10):1225-34. doi: 10.1016/j.chembiol.2013.07.016. Epub 2013 Sep 12.
In the actinorhodin type II polyketide synthase, the first polyketide modification is a regiospecific C9-carbonyl reduction, catalyzed by the ketoreductase (actKR). Our previous studies identified the actKR 94-PGG-96 motif as a determinant of stereospecificity. The molecular basis for reduction regiospecificity is, however, not well understood. In this study, we examined the activities of 20 actKR mutants through a combination of kinetic studies, PKS reconstitution, and structural analyses. Residues have been identified that are necessary for substrate interaction, and these observations have suggested a structural model for this reaction. Polyketides dock at the KR surface and are steered into the enzyme pocket where C7-C12 cyclization is mediated by the KR before C9-ketoreduction can occur. These molecular features can potentially serve as engineering targets for the biosynthesis of novel, reduced polyketides.
在放线紫红素II型聚酮合酶中，首个聚酮修饰是区域特异性的C9-羰基还原反应，由酮还原酶（actKR）催化。我们之前的研究确定actKR的94-PGG-96基序是立体特异性的决定因素。然而，还原区域特异性的分子基础尚未得到很好的理解。在本研究中，我们通过动力学研究、聚酮合酶重组和结构分析相结合的方法，检测了20个actKR突变体的活性。已鉴定出对底物相互作用至关重要的残基，这些观察结果提示了该反应的结构模型。聚酮类化合物停靠在酮还原酶表面，并被引导至酶口袋中，在C9-酮还原发生之前，C7-C12环化由酮还原酶介导。这些分子特征有可能成为新型还原聚酮类化合物生物合成的工程靶点。
