Collaborative Research: Molecular Design of Thermophilic Alcohol Dehydrogenase for Chiral Synthesis

本合作项目将对热厌氧菌乙醇二次醇脱氢酶（TeSADH）的生物化学和分子生物学进行研究。该酶具有一种新的代谢生物化学特性，从生理上讲，它由乙酰辅酶a经过两个还原步骤合成乙醇。这在一定程度上解释了其天然的广泛底物特异性。在本项目中，将使用TeSADH作为模型系统来了解酶的底物特异性和立体特异性的物理化学基础。TeSADH的基因工程研究将包括：1)合理设计底物结合小袋，以确定其在(S)-与(R)-对映体特异性中的作用；2)通过定向进化将TeSADH从(S)特异性酶进化为具有高(R)对映体特异性的酶；3)改变TeSADH辅助因子从NADP+向NAD+的特异性。TeSADH特别适合这些研究，因为它具有高热稳定性和广泛的底物特异性。突变的影响将根据底物特异性、动力学参数和对映体特异性进行评估。更广泛的影响：本研究所得的改性酶可能用于商业合成的手性中间体的制备。拟议的研究结果可能对更好地理解酶立体特异性的物理生化基础作出重大的智力贡献，并提供具有潜在工业用途的新型生物催化剂。该项目的外展计划包括培训多学科研究生和本科生，包括少数民族学生；在受资助院校的课程中传播知识；并在国家化学、生物化学、化学工程和微生物学会议上发表论文。

In this collaborative project, the biochemistry and molecular biology of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) will be investigated. This enzyme has a novel metabolic biochemistry, since physiologically it synthesizes ethanol in two reductive steps from acetyl-CoA. This, in part, explains its natural broad substrate specificity. In this project, TeSADH will be used as a model system to understand the physicochemical bases for substrate specificity and stereospecificity of enzymes. Genetic engineering studies of TeSADH will include: 1) Rational design of the small substrate-binding pocket to determine its role in (S)- versus (R)-enantiospecificity; 2) Evolving TeSADH by directed evolution from an (S)-specific enzyme into an enzyme with high (R)-enantiospecificity; and 3) Changing TeSADH cofactor specificity from NADP+ towards NAD+. TeSADH is uniquely suited for these investigations because of its high thermostability and its broad substrate specificity. The effects of the mutations will be evaluated in terms of substrate specificity, kinetic parameters, and enantiomeric specificity. Broader Impacts: The modified enzymes resulting from this research could be potentially used in preparation of chiral intermediates in commercial synthesis. The results of the proposed research are likely to make a significant intellectual contribution to a greater understanding or the physical biochemical basis of enzymatic stereospecificity, as well as to provide novel biocatalysts with potential industrial utility. The project's outreach program includes training multidisciplinary graduate and undergraduate students, including minorities; disseminating knowledge in courses at the grantee institutions; and presenting papers at national chemistry, biochemistry, chemical engineering, and microbiology meetings.