聚酮是自然界中的一大类活性多样的次级代谢产物，是临床药物的重要来源。其明显的结构特征是具有多个羟基和甲基手性中心。I型聚酮的生物合成研究发现，聚酮合酶模块中的酮基还原酶（KR）结构域不仅可以把β-酮基特异性地还原为D-或L-型羟基，还可以把D-型α-甲基异构化为L-型，这是聚酮结构多样性的重要原因。我们前期的工作确定了KR催化中心影响立体化学特异性的几个关键氨基酸残基，但具体机制仍不清楚。本研究中，我们将综合利用结构生物学、生物化学、分子生物学以及生物信息学等手段系统探索KR立体化学特异性的分子机制。利用X射线晶体衍射技术解析KR和底物复合物的三维结构，确定催化中心参与底物结合的氨基酸残基；利用定点突变和体外功能分析验证其对KR立体化学特异性的影响；结晶KR突变体，分析催化中心的结构变化；最后，利用所得到的结果指导KR立体化学特异性的定向改造，在微生物体内合成具有预期手性中心的聚酮化合物。

Polyketides are a large group of secondary metabolites with diverse bioactivities of which many have been clinically used as drugs. The presence of numerous methyl- and hydroxyl-bearing chiral centers is one of the most striking features of polyketides. During the biosynthesis of type I polyketides, a ketoreductase (KR) domain of a polyketide synthase module can stereospecifically reduce a β-keto to a D- or L-β-hydroxyl and epimerize a D-α-methyl to an L-α-methyl. Although several key residues that affect the stereochemical specificity of KR domains have been identified in our previous research, the detailed mechanism is still unclear. In this proposal, we will use a combination of structural biology, biochemistry, molecular biology, and bioinformatics approaches to elucidate the molecular basis of the stereochemical specificity of KR domains. Special emphasis will be placed on 1) solving the crystal structures of KR complexed with substrate by X-ray crystallography to identify residues involved in substrate binding; 2）elucidating the effects of these residues on stereochemical specificity of KR by site-directed mutagenesis and in vitro functional assay; 3) solving the crystal structures of KR mutants to reveal the conformation change of active site; and 4) generating polyketides with expected chiral centers in vivo using suitable microbial hosts by engineering KR stereochemical specificity with the resulting information.