2R,3R-丁二醇脱氢酶识别催化手性底物的结构和功能研究

2,3-Butanediol (2,3-BD) and acetoin (AC) regarded as C4 platform chemical, their biosynthesis is a hot spot of scientific researches. 2,3 Butanediol dehydrogenase (2,3-BDH) stereospecifically catalyzes the redox reaction between 2,3-BD and AC, and understanding of its molecular mechanism is essential for their biosynthesis. At present, although crystal structures of (2S,3S)-BDH and meso-BDH monomers and their inhibitor binding complexes were solved, due to the lack of (2R,3R)-BDH structural information, the mechanism for chiral substrate recognition by 2,3-BDH still remain unclear. To establish how 2,3-BDH possesses differential stereospecificities, we select (2R,3R)-BDH from Bacillus subtilis, crystalize and determine the X-ray crystal structures of (2R,3R)-BDH with chiral substrate complex. Combining with molecular simulation method such as molecular docking, structural analysis of (2R,3R)-BDH will discover the roles of catalytic residues on determining substrate stereospecificity. (2R,3R)-BDH mutants are created by site-direct mutagenesis of catalytic residues. Further structural analysis of the crystallized (2R,3R)-BDH mutants will help to clarify the subtle structural differences at the substrate-binding site like charge, polarity, hydrophobicity, and side chain conformations, and this will deepen our understanding of 2,3-BDH. Explaining the mechanism of chiral recognition of 2,3-BDH at the atomic level will enable further rational engineering of 2,3-BDH to change the topology of the substrate-binding site and adjust substrate stereoselectivity.

2,3-丁二醇和乙偶姻的生物合成是碳4平台化合物研究的热点，其核心科学问题是2,3-丁二醇脱氢酶（2,3-BDH）与手性底物特异识别及催化的分子机理。目前，虽然解析了(2S,3S)-BDH和meso-BDH单体和结合抑制剂的晶体结构，但是由于缺乏(2R,3R)-BDH的结构信息，导致2,3-BDH如何识别手性底物的分子机理不明确。本项目将通过解析枯草芽孢杆菌（Bacillus subtilis）来源的(2R,3R)-BDH及其同手性底物复合物的X射线晶体结构，结合分子对接的分子模拟手段，确定(2R,3R)-BDH识别和催化手性底物的关键氨基酸残基。在此基础上，通过重组表达突变体，对比研究(2R,3R)-BDH与突变体之间的构象差异、酶学性质不同，明确关键氨基酸残基的性质（电荷、极性、疏水和侧链构象）在识别和催化手性底物中的作用，为实现2,3-BDH专一高效催化手性底物的功能改造奠定基础。

2,3-丁二醇和乙偶姻的生物合成是碳4平台化合物研究的热点，其核心科学问题是2,3-丁二醇脱氢酶（2,3-BDH）与手性底物特异识别及催化的分子机理。目前，虽然解析了(2S,3S)-BDH和meso-BDH单体和结合抑制剂的晶体结构，但是由于缺乏(2R,3R)-BDH的结构信息，导致2,3-BDH如何识别手性底物的分子机理不明确。本项目通过解析枯草芽孢杆菌（Bacillus subtilis）来源的(2R,3R)-BDH及其同手性底物复合物的X射线晶体结构，结合分子对接的分子模拟手段，确定了(2R,3R)-BDH识别和催化手性底物的关键氨基酸残基。在此基础上，通过重组表达突变体，对比研究(2R,3R)-BDH与突变体之间的构象差异、酶学性质，明确关键氨基酸残基的性质（电荷、极性、疏水和侧链构象）在识别和催化手性底物中的作用，为实现2,3-BDH专一高效催化手性底物的功能改造奠定基础。