Bacterial P450 engineering for production of valuable drug metabolites

美国食品和药物管理局(FDA)建议，许多人类药物代谢物(以及它们的母体药物)应该接受安全/毒性测试。人类药物代谢中的主要酶是细胞色素P450，它催化许多人类药物和其他外源物质的氧化代谢。然而，由于人类P450及其还原酶是相对缓慢和不稳定的膜结合酶，它们不太可能用于药物代谢物的生产。同样，复杂药物代谢物的化学合成是费时费力的，药物的化学氧化很可能是非特异性的，除了所需的任何“人型”代谢物外，还会产生各种副产品。这个博士项目将通过使用一种高活性的、可溶的细菌P450酶(来自巨大芽孢杆菌的P450 BM3-一种高效的P450-P450还原酶融合酶)来解决这个问题。BM3是一种被广泛研究的酶，其催化活性是所有P450酶中最高的(对花生四烯酸氧化的催化活性为285 S-1)。我们最近的工作产生了BM3的“守门人”变体，其中BM3的S底物选择性图谱与野生型酶的选择性发生了显着变化，这是因为突变引入了更大的P450构象灵活性和更大的接近血红素的活性位点体积。在支持工作中，我们已经证明这些看门人突变体可以结合/代谢许多野生型BM3不能识别的底物。这些药物包括抗糖尿病药物曲格列酮和胃质子泵抑制剂奥美拉唑，以及类固醇(如睾酮)。重要的是，许多BM3代谢物与人类主要药物代谢P450的代谢物是相同的。这表明，高效的BM3守门人突变体可能为解决如何生产有效数量的人用药物代谢物进行药物安全性测试提供了一条重要的途径。该项目将在曼彻斯特的MIB为学生提供广泛的培训(重点是分子生物学；蛋白质表达/工程；化合物筛选和新药物底物的鉴定；酶周转和分析研究(使用核磁共振、高效液相色谱和GC-MS)来表征通过高通量化合物筛选确定的新底物形成的氧化产物；以及使用X射线衍射方法以新的底物结合形式的BM3守门人突变结晶)。在初步工作中，我们已经确定了BM3守门人突变体的新底物，方法是滴定BM3守门人突变体的潜在底物，并鉴定高自旋P450血红素铁物种的形成--这是一种典型的方法，用于识别新的P450底物并(通过UV-Vis光谱滴定)定量它们的亲和力(Kd值)。Cypex有限公司的研究将集中在BM3突变表达细胞的发酵，以促进药物进入，以及识别和量化形成的关键药物代谢物的分析研究。该项目将为学生提供广泛的酶学、结构生物学、微生物学和分析技术方面的培训，并将使学生掌握与在工业或学术界的职业生涯相关的重要技能。该项目与BBSRC在优先领域的职责保持一致，如“生物科学技术开发”(通过开发用于底物鉴定和鉴定的高通量筛网)和“工业生物技术和生物能源”(通过鉴定和鉴定由BM3看门人突变体以高产量产生的人类药物代谢物)。该项目还与DTP的“世界级生物科学基础”主题相一致(通过关键研究，开发使用BM3突变体生产真正的人类药物代谢物的有效途径，最终目的是生产大量这些代谢物，用于药物安全测试)。

The US Food and Drug Administration (FDA) have recommended that many human drug metabolites (as well as their parent drugs) should be subject to testing for safety/toxicity. The primary enzymes in human drug metabolism are the cytochromes P450, which catalyze oxidative metabolism of numerous human drugs and other xenobiotics. However, since human P450s and their reductases are relatively slow and unstable membrane-bound enzymes, they are unlikely to be useful in pharmaceutical metabolite production. Similarly, chemical synthesis of complex drug metabolites is laborious and the chemical oxidation of drugs is likely to be non-specific and to produce various side-products in addition to any of the desired "human-type" metabolite. This PhD project will address this problem by use of a high-activity, soluble bacterial P450 enzyme (P450 BM3 from Bacillus megaterium - an efficient P450-P450 reductase fusion enzyme). BM3 is an intensively studied enzyme due to its catalytic proficiency, and has the highest catalytic activity of any P450 enzyme (285 s-1 for oxidation of arachidonic acid). Our recent work has produced BM3 "gatekeeper" variants in which BM3's substrate selectivity profile is dramatically altered from that of the wild-type enzyme, due to mutations that introduce greater P450 conformational flexibility and a larger active site volume close to the heme. In underpinning work, we have shown that these gatekeeper mutants can bind/metabolise numerous substrates that are not recognized by wild-type BM3. These include pharmaceuticals such as the anti-diabetic troglitazone and the gastric proton pump inhibitor omeprazole, as well as steroids (e.g. testosterone). Importantly, many of the BM3 metabolites made are the same as those from the main human drug metabolising P450s. This shows that the highly efficient BM3 gatekeeper mutants could provide an important route to solving the issue of how to produce human drug metabolites in useful amounts for drug safety testing. This project will offer the student extensive training at the MIB in Manchester (focusing on molecular biology; protein expression/engineering; compound screening and identification of new drug substrates; enzyme turnover and analytical studies (using NMR, HPLC- and GC-MS) to characterize oxidized products formed from novel substrates identified by high-throughput compound screening; and BM3 gatekeeper mutant crystallization in novel substrate-bound forms using X-ray diffraction methods). In preliminary work we have already identified new substrates for the BM3 gatekeeper mutants by titrating the potential substrates against BM3 gatekeeper mutants and identifying the formation of a high-spin P450 heme iron species - a typical assay used to identify new P450 substrates and to quantify (by UV-vis spectroscopic titration) their affinity (Kd value). Studies at Cypex Ltd will focus on fermentation of BM3 mutant expression cells permeabilised to facilitate drug entry, and analytical studies to identify and quantify key drug metabolites formed. The project will provide the student with broad training in enzymology, structural biology, microbiology and analytical techniques, and will also equip the student with important skills relevant for a career in industry or academia. The project aligns with BBSRC remit in priority areas such as "Technology Development for the Biosciences" (through development of high-throughput screens for substrate identification and characterization) and "Industrial Biotechnology and Bioenergy" (through work to identify and characterize human drug metabolites produced in good yield by BM3 gatekeeper mutants). The project also aligns with the DTP theme of "World Class Underpinning Biosciences" (via key research to develop efficient routes to production of bona fide human drug metabolites using BM3 mutants, with the ultimate aim of producing large amounts of these metabolites for applications including drug safety testing).