Bacterial P450 engineering for production of valuable drug metabolites

美国食品药品监督管理局（FDA）建议，许多人用药物代谢物（以及其母体药物）应接受安全性/毒性测试。人类药物代谢中的主要酶是细胞色素P450，其催化许多人类药物和其它异生物质的氧化代谢。然而，由于人类P450及其还原酶是相对缓慢且不稳定的膜结合酶，因此它们不太可能用于药物代谢产物的生产。类似地，复杂药物代谢物的化学合成是费力的，并且药物的化学氧化可能是非特异性的，并且除了任何所需的“人类型”代谢物之外还产生各种副产物。这个博士项目将通过使用高活性，可溶性细菌P450酶（来自巨大芽孢杆菌的P450 BM 3-一种有效的P450-P450还原酶融合酶）来解决这个问题。BM 3是一种被深入研究的酶，由于其催化能力，并具有任何P450酶的最高催化活性（285 s-1的花生四烯酸氧化）。我们最近的工作已经产生了BM 3的“看门人”的变种，其中BM 3的底物选择性的配置文件是显着改变的野生型酶，由于突变，引入更大的P450构象的灵活性和更大的活性位点体积接近血红素。在基础工作中，我们已经证明这些看门突变体可以结合/代谢许多不被野生型BM 3识别的底物。这些包括药物，如抗糖尿病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，所产生的许多BM 3代谢物与主要人类药物代谢P450的代谢物相同。这表明，高效的BM 3看门突变体可以为解决如何产生用于药物安全性测试的有用量的人类药物代谢物的问题提供重要途径。该项目将在曼彻斯特的MIB为学生提供广泛的培训（侧重于分子生物学;蛋白质表达/工程;化合物筛选和新药底物的鉴定;酶周转和分析研究（使用NMR，HPLC和GC-MS），以表征通过高通量化合物筛选鉴定的新型底物形成的氧化产物;和BM 3看门突变体以新的底物结合形式结晶（使用X射线衍射方法）。在初步工作中，我们已经通过滴定针对BM 3看门突变体的潜在底物并鉴定高自旋P450血红素铁物质的形成来鉴定BM 3看门突变体的新底物-一种用于鉴定新P450底物并定量（通过紫外-可见光谱滴定）其亲和力（Kd值）的典型测定。Cypex Ltd的研究将集中在BM 3突变表达细胞的发酵，透化以促进药物进入，以及分析研究以鉴定和定量形成的关键药物代谢产物。该项目将为学生提供酶学，结构生物学，微生物学和分析技术方面的广泛培训，并将为学生提供与工业或学术界职业相关的重要技能。该项目与BBSRC在优先领域的职权范围保持一致，如“生物科学技术开发”（通过开发高通量筛选底物鉴定和表征）和“工业生物技术和生物能源”（通过鉴定和表征由BM 3看门突变体产生的高产人类药物代谢物）。该项目还与DTP主题“世界级基础生物科学”保持一致（通过关键研究开发使用BM 3突变体生产真正的人类药物代谢物的有效途径，最终目标是生产大量这些代谢物用于药物安全性测试等应用）。

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).