Biocatalytic Use of Cytochrome P450 Enzymes

描述（由申请人提供）：本提案的重点是扩大细胞色素P450单加氧酶变体的生物催化用途，包括芳香底物的硝化和脱硝，迄今尚未报道。具体来说，本研究将使用P450-BM3 (BM3)，因为它已被重新设计以结合和转化各种非生物底物。本研究的具体目标是：(1)发展和进化现有BM3变异体，利用活性氮对苯酚进行硝化；(2)优化BM3介导硝化作用的区域选择性、产物分布和底物范围；(3)发展和进化现有BM3变异体，对2-硝基苯酚起脱硝酶的作用；(4)扩大BM3变异体介导脱硝的底物和反应范围。最初，将对Arnold中现有的BM3文库进行硝化和脱硝化学筛选，并设计高通量比色筛选来快速测定新酶的活性。用于初始筛选的额外突变酶面板将使用关键残基的定点诱变，然后进行定向进化。随后的产物表征和优化变体的生化特性将用于制备第二代酶。最后，硝化和反硝化反应的底物范围将扩大到小分子（例如炸药、除草剂、荧光团）、药物和带有硝基官能团的生物分子。如有必要，可以对BM3酶进行进一步的定向进化，以优化底物结合和催化活性。这些目标的成功实现将为P450用于新化学物质的使用提供一个基本的见解，并将导致酶工具可用于合成应用，环境修复和诊断。

DESCRIPTION (provided by applicant): The focus of this proposal is to expand the biocatalytic use of cytochrome P450 monooxygenase variants to include nitration and denitration of aromatic substrates, which to date has not been reported. Specifically, P450-BM3 (BM3) will be used in this study since it has been re-engineered to bind and transform a variety of abiotic substrates. The specific aims for this proposal are to: (1) Develop and evolve existing BM3 variants to utilize reactive nitrogen species to nitrate phenol, (2) Optimize the regioselectivity, product distribution, and substrate scope of BM3 mediated nitration, (3) Develop and evolve existing BM3 variants to function as a denitrase on 2-nitrophenol, and (4) Expand the substrate and reaction scope of denitration mediated by BM3 variants. Initially, existing BM3 libraries in the Arnold will be screened for nitration and denitration chemistries, and high throughput colorimetric screens have been designed to rapidly assay new enzyme activity. Additional mutant enzyme panels for the initial screening will be generated using site directed mutagenesis of key residues followed by directed evolution. Subsequent characterization of the resulting products and biochemical properties of the optimized variants will be used to prepare second-generation enzymes. Finally, the substrate scope for both nitration and denitration reactions will be expanded to small molecules (e.g. explosives, herbicides, fluorophores), pharmaceuticals, and biomolecules bearing the nitro functional group. If necessary, BM3 enzymes can be subjected to further rounds of directed evolution to optimize substrate binding and catalytic activity. Successful accomplishment of these aims will provide a fundamental insight into the use of P450 for new chemistries and will result in enzymatic tools that can be used for synthetic applications, environmental remediation, and diagnostics.