醇脱氢酶对人工烟酰胺辅酶偏好性的分子机制研究

Biocatalytic asymmetric reduction employing alcohol reductases is a green and promising alternative for the synthesis of chiral alcohols. The reactions catalyzed by alcohol dehydrogenase requires NAD(P)H/NAD(P)+ to participate in proton transfer and energy metabolism. Since natural nicotinamide coenzymes are costly and instability, artificial coenzymes with stable performance and low cost have shown great application potential. At present, the molecular engineering on coenzyme preference of oxidoreductase is mostly for the switching of NADP(H) to NAD(H),whereas there is a lack of molecular engineering methods and theoretical guidance for artificial coenzyme preference. And also we have little knowledge yet for the catalytic mechanism of this oxidoreductase and artificial coenzyme interaction. The proposed project plans first to combined mutations of active sites in the coenzyme binding domain of newly mined alcohol dehydrogenase, KpADH from Kluveromyces polyspora, to reconstruct a hydrogen bond network, then variants which can use artificial coenzyme instead of NADPH for asymmetric reduction could be obtained by high-throughput screening. The interactions between residues and artificial coenzyme as well as the molecular mechanism of proton transfer are uncovered via analysis the structures of enzyme-coenzyme complex, thermodynamic parameters and computational simulation. The results will provide biocatalyst which is adapted to artificial coenzymes, and molecular basis for the molecular engineering of artificial coenzyme preference.

醇脱氢酶催化的不对称还原是合成手性醇最具应用潜力的绿色途径。醇脱氢酶驱动的反应需要烟酰胺类辅酶NAD(P)H/NAD(P)+参与进行质子及能量传递，由于天然辅酶价格昂贵、稳定性差，性能稳定且成本低廉的人工辅酶显示出极大的应用潜力。目前，烟酰胺辅酶依赖型氧化还原酶辅酶偏好性的改造集中于NADP(H)向NAD(H)转变，而针对人工辅酶相应酶的改造方法缺乏理论指导，二者的相互作用机制仍不清晰。本项目拟以前期获得的来源于Kluveromyces polyspora的醇脱氢酶KpADH为研究对象，通过对关键位点进行组合饱和突变重构辅酶结合域的氢键网络，筛选获得可利用人工辅酶进行羰基不对称还原反应的突变酶；通过晶体结构、热力学参数、计算机模拟揭示酶与人工辅酶的识别规律，阐明催化过程中的氢转移机制。本项目将提供一种可利用人工辅酶的突变酶，并对烟酰胺辅酶依赖型氧化还原酶人工辅酶偏好性改造提供理论指导。

氧化还原酶是第二大工业应用酶，也是目前已报道酶中数量最多的酶类。已知的氧化还原酶中约80%需要NAD(P)H作为辅酶，且催化时需要化学计量的辅酶参与反应。然而天然烟酰胺辅酶价格昂贵且化学性质不稳定，限制了氧化还原酶的工业应用。目前能够降低辅酶成本的方法有：一、构建辅酶循环再生系统；二、寻找结构简单、合成方便且化学性质稳定的人工辅酶来代替天然烟酰胺辅酶在生物催化体系中的应用。主要研究内容如下：.对野生型醇脱氢酶KpADH（WTKpADH）的辅酶结合域进行计算机模拟分析，结合烟酰胺辅因子偏好性改造工具CSR-SALAD确定KpADH辅酶偏好性改造热点S9、R32和K36，获得了对NADH和NADPH都具有较高利用能力的突变体S9N/R32C/K36I和专一性利用NADH的突变体S9A/R32C/K36I，其辅因子偏好性变化值分别是野生型的86.7和147.4倍。通过对位点E56、I57、S82、P83和P98饱和突变以恢复活力，筛选获得最佳突变体S9A/R32C/K36I/I57E催化活力从原来的0.160 U·mL-1提高到0.237 U·mL-1。.来自硫磺矿硫化叶菌的葡萄糖脱氢酶（Sulfolobus solfataricus glucose dehydrogenase, SsGDH)及其双突变体SsGDHI192T/V306I可依赖1,4-二氢吡啶（1,4-DHPs）类型的烟酰胺人工辅酶，包括：BNA+、P2NA+和P3NA+，催化葡萄糖的氧化，但对含有羧基官能团的人工辅酶BANA+。本研究以SsGDH为改造对象，通过分子对接分析获得SsGDH的突变热点E44和E114，对两个关键位点的饱和突变文库进行筛选，获得了对烟酰胺辅酶类似物利用能力提高的优势突变体获得了优势突变体E44N、E44D、E44W、E44Q、E114D和E114K，组合突变后获得最优的突变体E44D/E114L相比较野生型在以BANA+为辅酶进行催化时活力提高7.7倍，催化效率提高10.6倍。.