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 Our preliminary published work shows the viability of this plan (e.g. for omeprazole), and in unpublished work we have seen metabolites from a number of substrates (e.g. dextromethorphan) using conformationally perturbed BM3 mutant catalysts.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）具有至关重要的人类生理功能。负责性类固醇的几种关键氧化转化，并且是参与异生元解毒的主要酶。这些反应涉及O2对血红素铁的P450依赖性激活，并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（例如给出羟基化，尽管其他结果是可行的），而这种底物的化学氧化通常会导致几种不同的产品。人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。但是，在细菌中发现了较高的活性，可溶性P450。这些氧化酶最有效的催化效率是P450自然融合到NADPH依赖性还原酶。最好的研究是具有生物技术重要的P450 BM3，其营业额数量约为300/s，脂肪酸。我们通过其P450域的诱变来设计BM3，以促进构象重组。在这样做的产生的变体具有巨大变化的底物识别。这些包括在不同的突变特异性位置氧化类固醇的突变体；以及产生人类药物的代谢物的人，其主要人类P450催化剂与其主要的位置相同。学生训练的主要目标是利用BM3突变酶制造类固醇（孕酮，睾丸激素）和选定药物的氧化代谢产物。我们的初步发表的工作表明了该计划的生存能力（例如，对于奥美拉唑），在未发表的工作中，我们看到了使用构型扰动的BM3突变体催化剂的许多底物（例如脱氧核心）的代谢产物。学生将生成/净化BM3变体（包括A82F突变体）；此后，通过光学（血红素）滴定和稳态动力学来表征与一系列类固醇和药物的相互作用。与Agilent合作，将使用最先进的LC或GC-MS/MS设施分析与所有底物的氧化代谢产物，以鉴定氧化产物并量化其形成以及NADPH氧化与产品形成的程度。将确定产生药物/类固醇代谢产物（例如人代谢产物）的相关BM3突变体的血红素结构域的晶体结构，并确定与通过共结晶和/或浸泡配体无的晶体所寻求的底物的复合物。使用结构数据和/或分子建模包含预测的结合模式（基于药物/类固醇氧化的位置），将进行BM3合理工程，以提高所需的结合亲和力和/或氧化的区域选择性。在安捷伦（Agilent），使用纯突变酶的体外周转研究中的产物进行了关键的代谢物制备，将对第二代变体的产品曲线进行询问，并进行扩大研究，并将其与大肠杆菌转化剂提取物或整个细胞的效率进行比较。该学生将接受蛋白质工程，酶隔离/表征，光谱和结构生物学的培训；以及有机产品分析，定量和制备。该项目利用了学术和工业网站的补充专业知识，该学生将参与一个跨学科项目，该项目旨在旨在使BM3在制造可估计的人类类固醇/药物代谢物方面的重要合成生物学应用。