P450cam和P450BM3共进化同工酶的结构功能关系解析

细胞色素P450单加氧酶能够高效催化碳-氢键选择性氧化，在精细化学品及药物生产上具有巨大的应用价值。然而，利用（半）随机突变针对某一特定P450酶的非/半理性改造具有一定盲目性，缺少对P450酶家族共有底物识别与催化机理的规律性认识，极大限制了其更加广泛的应用。申请人前期工作中发现序列相似度低且天然底物谱截然不同的两种模式P450酶，即P450cam和P450BM3经共定向进化后可识别同一底物龙涎醚并催化相同反应（3α-羟化），该结果暗示不同P450酶可能存在一些隐藏的共有底物识别与反应模式。有鉴于此，本项目拟针对上述趋同进化获得的3α-羟化同工酶，研究不同活性口袋产生相同底物特异性和催化功能的结构基础，应用结构生物学探究参与底物识别/催化的关键氨基酸-结合口袋三维轮廓-立体/区域选择性之间相互关联规律，为快速理性改造P450酶提供新思路，同时为化学品和新药生产开发新型P450生物催化剂。

Cytochrome P450 monooxygenases can catalyze efficient and selective C-H bond oxidation, thus holding great application values in syntheses of drugs and fine chemicals. However, the use of (semi-)random mutagenesis strategies towards a specific P450 for non-/semi-rational engineering purposes via directed evolution is somewhat aimless. It remains a long-standing puzzle to understand the intrinsic laws for substrate recognition and catalytic mechanism in P450 superfamily. In our previous work, we found that co-directed evolution of the prototypic P450cam and P450BM3 with low sequence similarity and distinct substrate scopes led to a common substrate recognition (ambroxide) and catalytic activity (3α-hydroxylation). This result implicates that there might exist some intrinsic laws to guide the substrate recognition and reaction mode of P450 enzymes. Thus, the present project is aimed to understand how the distinct active sites of the two isoenzymes generate the same catalytic function from a structure biology angle. The interrelated concepts about the key active-site residues for substrate recognition and catalysis, 3-D contour of the buried cavity and regio-/stereoselectivity will be established in order to provide a deeper understanding of the structure-function relationship of P450 enzymes. This study will also facilitate the more efficient and precise protein engineering of cytochrome P450 enzymes in the future. The resulting mutant P450 enzymes could serve as useful biocatalysts for syntheses of chemicals and drugs.