Synthetic biology applications of P450 BM3

P450 BM3的合成生物学应用

• 批准号: 1621664
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1621664
• 关键词: Synthetic; biology; applications; P450; BM3

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 Our preliminary published work shows the viability of this plan (e.g. for omeprazole), and in unpublished work we have seen metabolites from a number of substrates (e.g. dextromethorphan) using conformationally perturbed BM3 mutant catalysts.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.

细胞色素P450（P450）具有重要的人类生理功能;负责性类固醇的几个关键氧化转化以及参与异生物质解毒的主要酶。这些反应涉及P450依赖性激活血红素铁上的O2和将氧原子插入底物中。这通常发生在给定底物上的特定位置（例如，产生羟基化，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伴侣相互作用。然而，在细菌中发现了更高活性的可溶性P450。这些氧化酶中催化效率最高的是与NADPH依赖性还原酶天然融合的P450。研究得最好的是具有生物技术重要性的P450 BM 3，其脂肪酸周转数为~300/s。我们已经通过诱变其P450结构域来工程化BM 3以促进构象重组;并且在这样做的过程中产生了具有显著改变的底物识别的变体。这些突变体包括在不同的、特定的位置氧化类固醇的突变体;以及产生在与其主要的人类P450催化剂相同的位置氧化的人类药物的代谢物的突变体。学生的主要目标是利用BM 3突变酶来制造类固醇（孕酮，睾酮）和选定药物的氧化代谢物。我们初步发表的工作显示了该计划的可行性（例如奥美拉唑），在未发表的工作中，我们已经看到了使用构象扰动的BM 3突变催化剂的许多底物（例如美沙芬）的代谢产物。学生将生成/纯化BM 3变体（包括A82 F突变体）;然后通过光学（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。与安捷伦合作，将使用最先进的LC或GC-MS/MS设施分析所有底物的氧化代谢产物，以鉴定氧化产物并定量其形成以及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（例如人代谢物）的相关BM 3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寻找与底物的复合物。使用包含预测结合模式的结构数据和/或分子建模（基于药物/类固醇氧化的位置），将进行BM 3合理工程化以根据需要改善结合亲和力和/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，并使用纯突变酶体外周转研究的产品进行关键代谢物制备的规模扩大研究，并将效率与E.大肠杆菌提取物或全细胞。学生将接受蛋白质工程，酶分离/表征，光谱学和结构生物学的培训;以及有机产品分析，定量和制备。该项目利用学术和工业场所的互补专业知识，学生将参与一个跨学科项目，旨在研究BM 3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。