洛伐他汀酯酶PcEST的催化机理及分子改造研究

As one of the main cholesterol-lowering drugs for hypercholesterolemia, simvastatin is industrially manufactured from lovastatin, a fungal polyketide produced by Aspergillus terreus, through two chemical steps: alkaline hydrolysis of lovastatin to produce monacolin J, and acylation of monacolin J to simvastatin using 2,2-dimethyl butyryl chloride. But this chemical method is high cost, low yield and serious pollution. Recently, a biocatalysis process for monacolin J conversion to simvastatin has been developed using acyltransferase LovD9. However, the very low activities of all existing lovastatin esterases to produce monacolin J seriously restrict the biosynthesis of simvastatin..Our previous works have firstly indicated that PcEST esterase from Penicillium chrysogenum has obvious industrial application prospect because of the nearly 232 times higher hydrolytic activity for lovastatin side chains than that of the patent reported EcBla4. However, there are still a lot of problems, such as the low level of soluble expression and stability of PcEST and the wide improvement space on its activity. And the limited knowledge on the relationship between structure and function and on the catalytic mechanism of all existing lovastatin esterases seriously limited the further engineering on PcEST to meet industrial applications. In this proposal, based on the previously solved PcEST and three PcEST-ligand complex structures, we aim to intense research the relationship between structure and function of PcEST, and then clarify its catalytic mechanism by using X-ray crystallography, mass spectroscopy, isotope labelling and other several biochemical methods. These studies will further provide guidance for semi-rational design of PcEST to achieve superiority mutant enzymes with better solubility, higher stability and activity, and finally promote the biosynthesis of simvastatin.

辛伐他汀作为主要的降血脂药，其工业生产主要以土曲霉发酵产物洛伐他汀为原料，经侧链碱水解和莫那可林J酰化化学合成得到，但该方法成本高、收率低且污染严重。目前已实现了酶催化代替化学法一步酰化莫那可林J生产辛伐他汀，但缺少高活性的洛伐他汀酯酶生产莫那可林J严重制约辛伐他汀的全生物合成。.我们前期首次发现产黄青霉PcEST蛋白特异性水解洛伐他汀的活性为专利报道EcBla4的232倍，极具工业应用前景。但其可溶表达率低、稳定性差且活性仍有较大提升空间，而目前还没有对洛伐他汀酯酶结构-功能关系和催化机制的研究，这极大限制了对PcEST的酶工程改造。本研究将在前期解析的PcEST酶、酶-配体复合物结构的基础上，借助晶体学、质谱学和同位素标记等多种生化手段，深入研究PcEST的结构-功能关系，阐明其催化机制；并基于此，半理性改造PcEST，筛选出可溶性好、稳定性强和高活性的突变酶，推进辛伐他汀的生物合成。

辛伐他汀是目前市场上主要的降血脂药物，Monacolin J是其工业合成的关键前体物。当前Monacolin J的工业生产工艺主要是通过碱水解洛伐他汀侧链获得，但该工艺繁琐，成本高且环境污染严重。使用高效特异的洛伐他汀水解酶进行酶促合成是绿色生产Monacolin J的理想策略之一，但缺少高效特异洛伐他汀水解酶是多年来Monacolin J酶法合成的关键瓶颈。课题组鉴定并表征了一个具有自主知识产权的高效特异洛伐他汀水解酶PcEST，其活性是现有专利报道水解酶的232倍，极具工业应用潜力，但其可溶表达和热稳定性仍待提升。.本项目通过解析PcEST、PcEST-Monacolin J、S57A（失活突变体）-洛伐他汀和PcEST-辛伐他汀复合物的4个高分辨率晶体结构，基于结构分析，同时结合突变、体外酶活、docking、ITC和质谱等多种实验手段，揭示了PcEST的Ser57-Tyr170-Lys60催化三联体及其周围的氢键网络，连同特异的底物结合通道共同决定了其水解洛伐他汀的高效性，而辛伐他汀侧链的位阻效应导致PcEST无法对其进行水解，保证了PcEST的特异性。在此基础上，项目组进一步提出了3个指示器帮助后续高效特异洛伐他汀酯酶的快速筛选，并尝试对PcEST进行了基于结构的理性改造，成功获得了可溶性表达和热稳定性明显改良的优势突变体D106A，这些研究成果一定程度推进了绿色工业生产Monacolin J的进程。