基于羰基还原酶ChKRED20晶体结构的双向底物步移

Reduction of prochiral ketones catalyzed by ketoreductases is one of the most efficient methods to obtain chiral alcohols. In the previous study, the ketoreductase ChKRED20 with independent intellectual property rights was obtained by our group, which followed anti-Prelog rule and preferred NADH to NADPH as the coenzyme. Most significantly, the ChKRED20 can utilize 2-propanol as a cheap co-substrate to regenerate reduced cofactor efficiently, with excellent activity. And we had successfully screened mutants of ChKRED20 with enhanced activity and thermostability through directed evolution strategy. In order to expand its application, we intend to further carry out molecular evolution based on the structure of the enzyme and enzyme/substrate complex. By analysis of the results from experiments and substrate simulation docking, the key amino acid residues related to the catalytic activity, substrate acceptance, stereoselectivity and stability of the enzyme, etc, will be disclosed. With modification on these hot pots, it aims to improve the ability of the enzyme to accept macroligand substrate, expanding the substrate spectrum. And on the other hand, shrink the substrate acceptance of the enzyme, retaining its ability of NADH-self cycling, which can be used as a tool for coenzyme cycling. By the strategy of substrate walking in two directions, a series of mutant enzymes will be obtained, which can enrich the functional base library. And the study on the relationship between protein structure and function will provide useful information for the rational design on other carbonyl reductase.

羰基还原酶生物催化是合成手性醇的重要方法之一。本课题组前期获得了自主知识产权的羰基还原酶ChKRED20，该酶遵循anti-Prelog规则，依赖NADH为辅酶，能以异丙醇为辅底物实现还原型辅酶的循环，具有良好的催化活性。经初步改造获得了耐热性和活力提高的突变子。为进一步拓展该酶的应用，我们拟对该酶进行深度改造。通过解析该酶及酶/底物复合物的晶体结构，深入了解催化功能域，以底物模拟对接等手段识别空间结构上与催化活力、底物接纳、立体选择性以及稳定性等相关的关键氨基酸残基，并对这些位点进行多轮改造。一方面，提高该酶接受大位阻底物的能力，拓展底物谱；另一方面，我们拟将该酶进行缩小底物谱的反方向进化，但保留其NADH自循环能力，将其发展成为辅酶循环的工具酶。本项目通过这种底物双向步移策略，将获得一系列突变体酶，丰富功能性基础酶库，同时蛋白结构与功能关系的研究也将为其他酶的理性设计提供有用信息。

羰基还原酶生物催化是合成手性醇的重要方法之一。自主知识产权的羰基还原酶ChKRED20具有良好的酶学性能。为了进一步拓展该酶的应用范围，我们基于该酶的晶体结构对其进行了理性/半理性的深度改造。首先，我们将羰基还原酶ChKRED20和ChKRED20/NAD+复合物进行结晶并解析了晶体结构。根据结构信息，以野生型酶不能催化的底物邻氯苯乙酮进行分子对接，利用丙氨酸扫描识别了关键位点H145A和M201A，结合底物催化口袋的出入口的改造，经多轮突变获得了一系列底物谱大幅度拓展的突变体酶（30余个），极大地拓展了该酶催化邻氯苯乙酮、2,4-二甲基苯乙酮等底物的能力。其次，通过重构底物结合口袋以扩大口袋体积和扩大底物/产物进出通道两个思路，识别了重要位点Q97L和S153L，并根据不同底物分子对接模型识别了其他位点，显著提高了该酶催化α-取代的苯乙酮类底物、α-及b-芳基酮酯类以及双芳基酮类等大位阻底物的能力。.另一方面，我们以反向思维减少底物结合口袋体积、增大位阻，获得了突变体I93H/G94L，该突变体缩小了催化底物范围但保留了异丙醇辅酶循环能力，通过偶联，使TtADH催化2-氧代-4-苯基丁酸乙酯的能力提高了1.5倍。.通过上述对ChKRED20酶的扩大和缩小底物范围的改造，实现了底物的双向步移。本项目不仅有效拓展了该酶的底物范围，获得一系列具有新的催化功能且有应用潜力的突变子，丰富了功能性基础酶库，而且获得的工具酶可以应用到需要NADH辅酶的偶联反应中。同时蛋白结构与功能关系的研究有助于进一步理解此类酶的催化机理，也将为其他羰基还原酶的理性设计提供新的视角。

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