醇脱氢酶立体选择性识别大位阻双芳基酮的分子机制研究

Chiral diaryl alcohols are important building blocks for the synthesis of pharmaceutically interesting molecules including antihistaminic and antidepressive drugs. The asymmetric reduction of the corresponding prochiral diaryl ketones is regarded as one of the most promising, green and efficient method for the synthesis of chiral diaryl alcohols. Due to the large steric hindrance of the two bulky aromatic side-chains of diaryl ketones, only a few biocatalysts have been reported. Therefore, the source of enzymes is extremely limited, and the biocatalytic reactions often suffer problems such as low substrate concentration, undesirable stereoselectivity, and limited substrate spectrum. In our previous study, a diaryl alcohol dehydrogenase KpADH (82%ee (R)) from Kluyveromyces polysporus was identified by genome mining. In this project, key residues involved in substrate binding will be predicted based on homology model of KpADH. Using the hydroclassified combinatorial saturation mutagenesis (HCSM) strategy we proposed, smart library of the above identified key residues will be constructed for the co-evolution of R-selectivity and activity. Additionally, the substrate binding pocket will be reshaped based on the hydrogen bond network to invert the stereoselectivity of KpADH from R to S. The evolutionary path of the key residues in stereoselectivity will be explored. Also, the crystal structures of wide-type KpADH, R-/S-selective mutants and their complex with substrate/cofactor will be resolved to elucidate the stereoselective recognition and catalysis mechanisms of bulky diaryl ketones. Based on the reaction kinetics, a highly efficient biocatalysis process with cofactor regeneration and green solvent system will be developed for the preparation of chiral diaryl alcohols.

手性双芳基醇是一类重要的砌块分子，可用于合成抗组胺过敏药、抗抑郁药等药物。生物不对称还原潜手性双芳基酮是合成手性双芳基醇的绿色高效方法。由于双芳基酮两侧的空间位阻较大，仅有少量生物还原法的报道，且存在酶来源有限，底物浓度低，立体选择性不理想等问题。本项目从基因组挖掘获得来源于多孢克鲁维酵母的双芳基醇脱氢酶KpADH（82%ee (R)）出发，基于同源建模确定底物结合关键位点，采用自主提出的亲疏水性组合饱和突变等策略构建聪明文库实现R-选择性和催化活性的共进化；基于氢键网络等特性进行底物结合口袋重构实现KpADH立体选择性从R到S的反转，探究关键氨基酸位点在决定其立体选择性中的进化路径；解析KpADH野生酶、突变酶及其与底物/辅酶复合物的晶体结构，阐明其立体选择性识别和催化大位阻双芳基酮底物的分子机制；基于偶联反应动力学、辅因子再生和介质系统优化，构建合成光学纯双芳基醇的高效生物催化体系。

光学纯(S)-(4-氯苯基)-(吡啶-2-基)甲醇[(S)-CPMA)是合成抗组胺贝托司汀类药物的重要手性中间体，由于底物酮的双芳基结构具有极大的空间位阻，只有少量醇脱氢酶被报道具有不对称催化还原双芳基酮的活力，因此它们通常被称为“难还原”的酮类。在前期工作中，采用基因挖掘的方法获得了来源于Kluyveromyces polyspora的NADPH依赖型短链醇脱氢酶KpADH，该酶催化还原CPMK时表现出较高的底物转化率和中等的立体选择性（82% ee, R）。.根据2,4-二硝基苯肼与羰基化合物的颜色反应特性，建立了醇脱氢酶催化还原活力的高通量筛选方法。CPMK为底物时，与DNPH反应生成产物在波长500 nm处具有特征吸收峰，摩尔消光系数为13750·L·mol-1·cm-1。以来源于Saccharomyces cerevisiae的NADPH依赖型甲基乙二醛还原酶GRE2为模板构建KpADH的同源模型，获得了KpADH底物结合口袋上16个氨基酸位点。选择天冬氨酸Asn和Val分别作为“极性筛子”和“非极性筛子”，得到6个与立体选择性相关的突变位点。采取“逐步优化组合策略”构建突变文库，获得立体选择性提高的突变体Mu-R2（99.2% ee，R）和立体选择性翻转的突变体Mu-S5（97.8% ee，S）。对WT KpADH及其突变体Mu-R2及Mu-S5进行蛋白质结晶，获得了这三个蛋白与NADPH的复合物晶体。分子动力学模拟结果显示Mu-R2与CPMKProR及Mu-S5与CPMKProS更易形成预反应状态。对于突变体Mu-S5，其底物结合口袋入口处突变位点N136，V161，C237和G214的α-碳原子可形成类平面的“极性门”。由于底物潜手性碳两侧氯苯环和吡啶环的带电差异，在催化过程中底物的取向在其穿过“极性门”时即被决定。对于野生型KpADH，类似的平面被周围芳香族氨基酸残基的侧链阻挡，从而使底物在接近催化中心时无法保持单一取向。建立双芳基醇脱氢酶KpADH和葡萄糖脱氢酶BmGDH偶联反应体系，将双酶共固定化实现双芳基手性醇(S)-CPMA的酶法连续合成。使用10 mL Ni-NTA对酶进行固定化，当流速为5 mL·min-1时反应1-3天底物转化率均为100%，反应时空产率为1560 g·L-1·d-1，最终产物得率为84.0%。

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