Y-内酰胺酶趋异进化的分子机制研究

It is a hot research field of molecular enzymology to reveal whether an enzyme molecule has catalytic promiscuity and clarify the divergent evolution mechanisms of promiscuous enzymes. Previously,a gene named gla from Microbacterium hydrocarbonoxydans was cloned and identifed to have two catalytic activities (Y-lactamase and perhydrolase) by bioinformatics analysis and substrate shift assay. In this study, gla is used as a model promiscuous gene to study the gene divergent evolution. Firstly, the system of random mutation and self-expression of the target gene is constructed in E. coli. After several rounds of high-throughput differential screening, the mutants with high specific activity of the lactamase and perhydrolase, respectively, as well as mutans with new feature of expoxide hydrolase activity could be obtained seccesfully.Further deconvolution experiments and enzymological characterization are performed to clarify the relationship of the changes in various enzyme activity during the divergent evolutionary pathway,and reveal the intrinsic link between the different point mutations and the different enzymatic functions such as activity, selectivity, stability, etc. The molecular dynamics simulations will be applied to reveal the dynamic foundations of enzyme function changes leaded by point mutations. This study will help to charify the divergent evolution mechanisms of such promiscuous enzyme, and the mutants with high activity and steroselectivity can be utilized as chiral biocatalysts to prepare the chiral drug intermediates.

揭示一个蛋白是否具有催化不专一性以及阐明多功能酶趋异进化的机制是当前分子酶学研究的热点领域。前期通过生物信息学分析和底物迁移实验，从氧化烃微杆菌中鉴定了一个同时具有Y-内酰胺酶、过水解酶活力的基因gla。本课题拟以gla为模型基因，首先在大肠杆菌中构建靶基因的随机自突变和自表达体系，通过多轮高通量差异筛选，分别获得高比活的内酰胺酶和过水解酶突变体，以及具有新功能的环氧化物水解酶突变体。进一步通过deconvolution 实验和详细的酶学表征，阐明gla基因趋异进化路径中各种酶活力变化的相互关系；揭示不同突变位点累积与不同功能（活性、选择性、稳定性等）变化的内在联系。通过分子动力学模拟分析揭示点突变导致酶功能变化的动力学基础。本研究不仅有助于阐明此类多功能酶趋异进化的分子机制，而且筛选获得的高活性和高立体选择性突变体可以用于制备重要的手性药物中间体。

酶趋异进化是一个高度复杂的现象，是生物多样性的重要分子基础。以Mhg为目标的研究过程中，我们通过一系列方法验证，最后发现底物通道是决定Mhg功能异变的重要结构基础，通过系列的突变分析发现底物通道入口处233位的关键氨基酸直接决定Mhg的功能分化，该位置不同氨基酸的取代可以分别出现酰胺酶、酯酶、溴过氧化物酶单活性突变体以及两两组合的活性突变体。此外，发现233位点不仅是Mhg酶活性异化的关键位点，同时是调节酶可溶性表达水平的关键位点，该位置上有些氨基酸的存在直接导致表达的蛋白全部以包涵体存在，而有些氨基酸则全部形成可溶性蛋都白，也发现一些氨基酸的存在包涵体和可溶性蛋白都有，且因氨基酸不同而比例不同。酶底物通道上存在关键氨基酸残基，同时决定酶的功能异变及蛋白的可溶性表达，这一重要的发现可以深化我们对酶的趋异进化的理解，也为通过进化改变蛋白在生物体内的可溶性表达提供了新的解决思路。