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

在这个合作项目中，将研究乙醇型嗜热厌氧杆菌次级乙醇脱氢酶(TeSADH)的生物化学和分子生物学。这种酶具有一种新的代谢生物化学，因为它在生理上通过两个还原步骤从乙酰辅酶A合成乙醇。这在一定程度上解释了其天然的广泛底物专一性。在这个项目中，TeSADH将被用作一个模型系统，以了解酶的底物专一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)合理设计小底物结合口袋，以确定其在(S)对(R)对映体专一性中的作用；2)通过定向进化将TeSADH从(S)专一性酶进化为具有高对映体专一性的酶；以及3)将TeSADH辅因子专一性从NADP+改变为NAD+。TeSADH具有很高的热稳定性和广泛的底物专一性，因此非常适合于这些研究。突变的影响将通过底物专一性、动力学参数和对映体专一性来评估。更广泛的影响：本研究得到的修饰酶有可能用于制备商业合成中的手性中间体。拟议的研究结果可能对更好地理解酶的立体专一性的物理生化基础做出重大的智力贡献，并提供具有潜在工业用途的新型生物催化剂。该项目的推广计划包括培训包括少数族裔在内的多学科研究生和本科生；在受资助机构的课程中传播知识；在全国化学、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.