以1,4-丁二醇为辅底物再生NADPH的醇脱氢酶的构建及催化机制研究

Biocatalytic asymmetric reduction employing alcohol reductases is a green and promising alternative for the synthesis of chiral secondary alcohol. The alcohol dehydrogenases harboring substrate-coupled cofactor regeneration system are industrially preferable due to its ability in elegant achievement the asymmetric reduction of prochiral ketone and cofactor regeneration in situ. Compared with isopropanol, 1,4-butanediol, having two free hydroxyl groups, could work as a ‘smart’ co-substrate for these alcohol dehydrogenases in the efficient and green regeneration of cofactor. However, only several alcohol dehydrogenases were reported with the ability in regeneration of NADH by oxidation of 1,4-butanediol and no NADPH-dependent dehydrogenases was discovered. And also we have little knowledge yet for the catalytic mechanism of this substrate-coupled cofactor regeneration. The proposed project plans first to investigate the relationship between co-substrate and residues in the binding pocket from newly mined alcohol dehydrogenase, KpADH from Kluveromyces polyspora, to get the hotspots for mutation. Through the high-throughput screening of the combinatorial saturation and DNA shuffling libraries of the hotspots, variants possessed the ability in regeneration of NADPH with 1,4-butanediol and retained the high enantioselectivity in the reduction of substrate are returned The interactions of residues and 1,4-butanediol and the molecular mechanism of NADPH regeneration are uncovered via analysis the structures of enzyme-substrate complex, reaction kinetics and spectroscopy data. The results will provide biocatalyst for efficient NADPH regeneration with 1,4-butanediol as co-substrate and the theoretical basis for the molecular engineering of the cofactor regeneration of the substrate-coupled alcohol dehydrogenase.

醇脱氢酶催化的不对称还原是合成手性仲醇最具应用潜力的绿色途径之一，具有底物偶联型辅因子再生系统的醇脱氢酶可便捷地实现底物的还原和辅因子在位再生，而备受工业青睐。相比异丙醇，1,4-丁二醇作为这类酶的“智能”辅底物因含两个羟基可实现辅因子高效、绿色再生。但仅少数酶可将其氧化以再生NADH，且无NADPH再生的报道，辅因子循环的机制也不明确，因此亟待研究和阐明。本项目拟以申请人前期获得的来源于Kluveromyces polyspora的醇脱氢酶KpADH为研究对象，系统研究参与辅底物结合的氨基酸位点；对结合位点进行组合饱和突变和DNA改组，高通量筛选获得兼具氧化1,4-丁二醇再生NADPH和高对映选择性的突变酶；结合晶体结构、反应动力学和光谱学结果，揭示酶与辅底物的识别规律，阐明催化机制。项目将提供一种以1,4-丁二醇为辅底物高效再生NADPH的工具酶，并为辅因子再生系统的改造提供理论指导。

氧化还原酶在精细化学品合成中的作用日趋重要，正逐渐由化学合成工艺的必要补充变为首要选择。采用酶法合成高附加值产品对于推动产业技术升级，实现国民经济新旧动能的转换具有重要意义。然而，氧化还原酶需要昂贵的辅酶进行电子传递，引入辅酶再生系统可显著降低成本。1,4-丁二醇作为一种智能辅底物，可避免添加过量辅底物和无副产物抑制。然而，目前可利用1,4-丁二醇再生NADPH的酶活力较低且分子机制不明确。本项目立足于构建高效利用1,4-丁二醇再生NADPH的辅酶再生系统，并解析该酶催化连续两步反应的分子动力学机制。. 围绕项目的研究目标，主要开展了四个方面的研究：（1）醇脱氢酶KpADH底物结合口袋氨基酸的定点饱和突变和迭代饱和突变；（2）KpADH蛋白质结晶和空间结构解析；（3）基于双底物动力学分析和分子动力学模拟解析反应机制；（4）利用1,4-丁二醇再生NADPH系统的构建。基于KpADH的结构模型，发现V84，Y127，S196，F197，V918和E214是参与1,4-丁二醇结合的关键位点。对上述位点进行迭代饱和突变，经高通量筛选获得突变体KpADHV84I和KpADHV84I/Y127M的比活力分别是野生型酶的3.9倍和2.7倍。动力学分析发现KpADHV84I/Y127M对1,4-丁二醇的催化专一性常数kcat/KM是野生型酶的12倍。底物特异性分析发现，野生型和突变型酶均对长链二醇表现出更高的催化效率。对两步反应动力学参数比较发现，两步反应的动力学参数差距非常大。与野生型相比，KpADHV84I/Y127M对两个底物的kcat/KM比例由6820降低至98。利用该突变酶构建了NADPH高效再生系统，并成功用于D-苯丙氨酸的合成。通过计算分析发现该反应遵循顺序bi bi机制，且两步反应的动力学匹配度对于该辅酶再生系统的高效运转具有重要的作用。解析KpADH的空间结构，并结合分子动力学模拟发现双突变酶对1,4-丁二醇具有更高的结合能。. 本项目基于自主开发的醇脱氢酶突变体揭示了其顺序氧化1,4-丁二醇再生NADPH的分子动力学规律及反应机制，对于深入了解醇脱氢酶催化机制和理性开发高效辅酶再生系统具有重要借鉴意义。