Synthetic biology applications of P450 BM3

细胞色素P450 （P450）具有重要的人体生理功能；负责性类固醇的几个关键氧化转化，以及参与外源解毒的主要酶。这些反应包括依赖于p450的O2在血红素铁上的激活和在底物中插入氧原子。这通常发生在给定底物的特定位置（例如羟基化，尽管其他结果也是可行的），而这些底物的化学氧化通常会产生几种不同的产物。人类p450是膜结合的，通常不稳定，并与膜相关的氧化还原伙伴相互作用。然而，在细菌中发现了活性更高的可溶性p450。这些氧化酶中催化效率最高的是自然融合到nadph依赖性还原酶的p450酶。研究得最好的是生物技术上重要的P450 BM3，它与脂肪酸的周转率为~300/s。我们通过诱变BM3的P450结构域来促进构象重组；在这样做的过程中，产生了具有显著改变底物识别的变体。这些突变包括在不同的突变特异性位置氧化类固醇的突变体；以及产生人类药物代谢物的基因，其氧化位置与主要的人类P450催化剂相同。该研究的主要目标是利用BM3突变酶制造类固醇（孕酮、睾酮）和选定药物的氧化代谢物。我们初步发表的工作表明了这一计划的可行性（例如奥美拉唑），在未发表的工作中，我们已经看到使用构象扰动的BM3突变催化剂从许多底物（例如右美沙芬）中产生代谢物。学生将生成/纯化BM3变体（包括A82F突变体）；此后，通过光学（血红素）滴定和稳态动力学表征与一系列类固醇和药物的相互作用。与Agilent合作，所有底物的氧化代谢产物将使用最先进的LC或GC-MS/MS设备进行分析，以识别氧化产物并量化其形成以及NADPH氧化与产物形成耦合的程度。将确定产生感兴趣的药物/类固醇代谢物（例如人类代谢物）的相关BM3突变体的血红素结构域的晶体结构，并通过共结晶和/或浸泡无配体晶体来确定与底物的配合物。利用结构数据和/或分子模型，包括预测的结合模式（基于药物/类固醇氧化的位置），BM3合理工程将根据需要提高结合亲和力和/或氧化的区域选择性。在安捷伦，将对第二代变体的产品概况进行调查，并对关键代谢物制备进行扩大研究，使用纯突变酶进行体外转化研究的产品，并与大肠杆菌转化提取物或全细胞的效率进行比较。该学生将接受蛋白质工程、酶分离/表征、光谱学和结构生物学方面的培训；以及有机产品的分析，定量和制备。该项目利用学术和工业领域的互补专业知识，该学生将参与一个跨学科项目，旨在研究BM3在制造有价值的人类类固醇/药物代谢物方面的重要合成生物学应用。

The cytochromes P450 (P450s) have crucial human physiological functions; being responsible for several key oxidative transformations of sex steroids as well as being primary enzymes involved in xenobiotic detoxification. These reactions involve P450-dependent activation of O2 on a heme iron and insertion of an oxygen atom into the substrate. This usually occurs at a specific position on a given substrate (e.g. giving hydroxylation, though other outcomes are feasible), whereas chemical oxidation of such substrates often results in several different products. The human P450s are membrane-bound, often unstable and interact with membrane-associated redox partners. However, higher activity, soluble P450s are found in bacteria. The catalytically most efficient of these oxidases are P450s naturally fused to a NADPH-dependent reductase. The best studied is the biotechnologically important P450 BM3 with turnover numbers of ~300/s with fatty acids. We have engineered BM3 by mutagenesis of its P450 domain to facilitate conformational reorganization; and in so doing produced variants with dramatically altered substrate recognition. These include mutants that oxidize steroids at distinct, mutant-specific positions; and ones generating metabolitesof human drugs oxidized in the same positions as done by their major human P450 catalysts. The main objectives of the studentship are to exploit BM3 mutant enzymes to make oxidized metabolites of steroids (progesterone, testosterone) and selected drugs. Our preliminary published work shows the viability of this plan (e.g. for omeprazole), and in unpublished work we have seen metabolites from a number of substrates (e.g. dextromethorphan) using conformationally perturbed BM3 mutant catalysts. The student will generate/purify BM3 variants (including A82F mutants); thereafter characterizing interactions with a range of steroids and pharmaceuticals by optical (heme) titrations and steady-state kinetics. In collaboration with Agilent, products of oxidative metabolism with all substrates will be analyzed using state-of-the-art LC or GC-MS/MS facilities to identify oxidized products and quantify their formation and the extent to which NADPH oxidation is coupled to product formation. Crystal structures of heme domains of relevant BM3 mutants that produce drug/steroid metabolites of interest (e.g. human metabolites) will be determined, andcomplexes with the substrates sought by co-crystallization and/or soaking of ligand-free crystals. Using structural data and/or molecular modelling embracing predicted binding mode (based on position of drug/steroid oxidation), BM3 rational engineering will be done to improve binding affinity and/or regioselectivity of oxidation as required. At Agilent, product profiles of 2nd generation variants will be interrogated and scale-up studies for key metabolite preparation done, using products from in vitro turnover studies with pure mutant enzymes, and comparing efficiency with that from E. coli transformant extracts or whole cells. The student will be trained in protein engineering, enzyme isolation/characterization, spectroscopy and structural biology; as well as in organic product analysis, quantification and preparation. The project exploits complementary expertise at the academic and industrial sites, and the student will engage in an interdisciplinary project aimed at important synthetic biology applications of BM3 in makingvaluable human steroid/drug metabolites.