Biocatalytic Use of Cytochrome P450 Enzymes

描述（由申请人提供）：该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，以包括芳香族底物的硝化和反硝化，迄今为止尚未报道。具体而言，P450-BM 3（BM 3）将用于本研究，因为它已被重新设计以结合和转化各种非生物底物。这项建议的具体目标是：（1）开发和进化现有的BM 3变体以利用活性氮物质来硝化苯酚，（2）优化BM 3介导的硝化的区域选择性、产物分布和底物范围，（3）开发和进化现有的BM 3变体以用作2-硝基苯酚的硝化酶，（4）扩大了BM 3变异体介导的酶解反应的底物和反应范围。最初，Arnold中现有的BM 3文库将被筛选用于硝化和反硝化化学，并且高通量比色筛选已被设计用于快速测定新的酶活性。将使用关键残基的定点诱变，然后进行定向进化，生成用于初始筛选的其他突变酶组。随后对所得产物的表征和优化变体的生物化学性质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展到小分子（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，BM 3酶可以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在新化学中的应用提供基本的见解，并将导致可用于合成应用，环境修复和诊断的酶工具。

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.