亮氨酸脱氢酶催化合成（S）-2-氨基-1-丁醇的新反应设计与催化机制研究

(S)-2-amnio-1-butanol, an important intermediate in the synthesis of antituberculous drug Ethambutol, is mainly synthesized by chemical or enzymatic resolution with limited applications due to environmental concerns or low conversion (max 50%). Toward green and efficient synthesis of (S)-2-amnio-1-butanol by new enzyme and reaction design and perform catalytic mechanism studies offer significantly to basic and application research. We have obtained a few mutants of leucine dehydrogenase which could catalyze the key intermediate 2-keto-1-butanol to (S)-2-amnio-1-butanol, but with lower catalytic efficiency as well as the unclearly catalytic mechanism. The new developed directed evolution strategy “Triple Code Saturation Mutagenesis (TCSM)” will be used for redesign and directed engineering the catalytic pocket of leucine dehydrogenase to improve and optimize the catalytic efficiency and stereoselectivity to 2-keto-1-butanol. Unveiling the catalytic mechanism of substrate specificity and chiral catalytic regulation related to substrate binding pocket reshaping of key mutants based on X-ray crystal structures resolution and molecular dynamic (MD) simulations in combination with quantum chemistry (QM) calculations, offering design principle and application guide to new enzyme and new reaction design.

（S）-2-氨基-1-丁醇是重要医药中间体，主要用来合成抗结核药盐酸乙胺丁醇。目前主要通过化学法和酶法拆分获得，但化学法污染大，路线长效率低；拆分法转化率受限于50%。因此，通过新酶新反应设计实现（S）-2-氨基丁醇的绿色高效合成并开展催化机制研究具有重要的科学研究与应用价值。申请人前期已经获得了可催化前体2-酮-1-丁醇为（S）-2-氨基丁醇的亮氨酸脱氢酶突变体，但其催化效率较低，而且催化机制尚不清楚。本项目拟采用三密码子饱和突变（TCSM）等技术方法对亮氨酸脱氢酶LeuDH的催化口袋进行再设计与定向改造，提高其对2-酮-1-丁醇的催化效率与立体选择性。并基于晶体结构解析、分子动力学模拟和量子化学计算等技术手段，解析关键突变体的底物选择与手性调控催化机制，揭示该酶底物催化结合口袋重塑设计与底物催化特异性及手性催化之间的适配规律，为同类型新酶与新反应设计提供理论和实践指导。

手性胺醇是一类重要的医药中间体砌块，例如（S）-2-氨基-1-丁醇是重要医药中间体，主要用来合成抗结核药盐酸乙胺丁醇。目前主要通过化学法和酶法拆分获得，但化学法污染大，路线长效率低；拆分法转化率受限于50%。因此，通过新酶新反应设计实现（S）-2-氨基丁醇的绿色高效合成并开展催化机制研究具有重要的科学研究与应用价值。项目资助期间，申请人构建了基于荧光分子探针的高通量筛选酮醇类化合物的检测方法，并将其应用于工程化胺脱氢酶的筛选，成功获得多种对2-氨基丁酮底物有活性目的基因。再此基础上，采用三密码子饱和突变（TCSM）等技术方法对工程化胺脱氢酶的催化口袋进行再设计与定向改造，进一步提高催化效率（转化率>99%, (S)-选择性ee值>99%）。最后通过计算模拟技术，解析关键突变体的底物选择与手性调控催化机制，揭示该酶底物催化结合口袋重塑设计与底物催化特异性及手性催化之间的适配规律，为同类型新酶与新反应设计提供理论和实践指导。