Evolution of efficient p450 biocatalysts for human drug metabolite production

用于人类药物代谢物生产的高效 p450 生物催化剂的演变

• 批准号: BB/G016968/1
• 金额: $ 9.48万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG016968%2F1
• 关键词: Evolution; efficient; p450; biocatalysts; human

Human cytochromes P450 (P450s) are essential enzymes in metabolism of drugs/xenobiotics entering the body, and for generation of numerous steroids and lipid mediators. P450s catalyse oxidative metabolism of their substrates, often with exquisite regio- and stereoselectivity of oxidation. There is great interest in application of P450s for production of oxyfunctionalized molecules as e.g. intermediates in chemical synthesis or high-value lipid mediators with specific biological functions (e.g. derivatives of arachidonic acid), and these molecules are often extremely difficult, inefficient and non-cost-effective to produce by organic synthesis. Of particular value in the human P450 field would be a ready source of key metabolites produced from a cluster of the P450s known to be most important for metabolism of prescribed drugs and pharmaceuticals (specifically CYP3A43, CYP2D6, CYP2C9, CYP2C19 and CYP1A2, which collectively oxidise and metabolise >90 % of administered drugs). These metabolites would be of great value in diagnostics for human P450 function (i.e. as standards for human P450 and hepatic metabolism) and as reagents that will e.g. help define variant/polymorphic P450 activity (and hence likelihood of adverse drug reactions) and enable quantification of key metabolite production. The problem is that the relevant human P450s and their diflavin P450 reductase partner are integral membrane proteins, are unstable and have low activities. Hence yields of relevant products are very small and a commercial process that exploits these enzymes is not financially viable. Instead, we will use the highest activity P450 in the entire P450 superfamily (P450 BM3 from Bacillus megaterium) for this purpose, and evolve BM3 to catalyse specific functions by a combination of rational mutagenesis and directed evolution, exploiting robotic facilities for screening provided already by BBSRC funding. The major reasons for exploiting BM3 are that a) it is a natural and soluble fusion of a P450 to its P450 reductase, with catalytic activity typically ~1000-fold that of the multi-component human enzymes, and b) it has already been shown (through work in our own labs and elsewhere) that the active site of the enzyme is pliable and can be engineered to bind and oxidise molecules quite different from its 'natural' fatty acid substrates (e.g. aromatics and polycyclic aromatics, short chain alkenes and alkanes). The student will undertake a combination of rational mutagenesis and directed evolution (with rational targets including Phe87, Val78, Ala264 and Leu181, which we have shown critical to control of substrate selectivity) in order to diversify reactivity of BM3 such that it will oxidise prototypical substrates of the aforementioned human P450s: testosterone (3A4), dextramethorphan (2D6), diclofenac (2C9), diazepam (2C19) and phenacetin (1A2). Our work has already shown that BM3 oxidises testosterone (but not at correct carbon for wild-type BM3), and all other human P450 substrates targeted have molecular dimensions and chemical characteristics similar to other compounds oxidised by various BM3 mutants. We will screen for activities towards new substrates using robotics and E. coli cell extracts expressing BM3 mutants, and oxo-plate technology whereby dye-linked oxygen consumption is measured. Point mutants that enhance activity with key substrates will be combined with variants randomly evolved in regions known to control substrate selectivity (in I helix, F/G and B/C loop regions) and improved mutants identified by further screening. Organic products will be characterized by GCMS/HPLC methods, and optimised enzymes purified and characterized enzymatically and structurally (in ligand-free and substrate-bound forms). The student will be supported by a PDRA working on a parallel P450 evolution project, and will develop novel catalysts for generation of important molecules to be exploited as reagents and diagnostics for commercial exploitation.

人细胞色素P450（P450）是进入体内的药物/异生物质代谢中的必需酶，并且用于产生许多类固醇和脂质介质。P450催化其底物的氧化代谢，通常具有精确的氧化区域和立体选择性。P450用于生产氧官能化分子作为例如化学合成中的中间体或具有特定生物学功能的高价值脂质介质（例如花生四烯酸的衍生物）的应用引起了极大的兴趣，并且这些分子通过有机合成生产通常是极其困难、低效且不具成本效益的。在人P450领域中特别有价值的是由已知对处方药物和药物的代谢最重要的P450簇（特别是CYP 3A 43、CYP 2D 6、CYP 2C 9、CYP 2C 19和CYP 1A 2，它们共同氧化和代谢> 90%的施用药物）产生的关键代谢物的现成来源。这些代谢物将在人P450功能的诊断中具有很大价值（即作为人P450和肝代谢的标准品），并且作为试剂，其将例如帮助定义变体/多态性P450活性（以及因此药物不良反应的可能性），并能够量化关键代谢物的产生。问题是相关的人类P450和它们的二黄素P450还原酶伴侣是整合的膜蛋白，不稳定并且具有低活性。因此，相关产品的产量非常小，利用这些酶的商业方法在经济上是不可行的。相反，我们将使用整个P450超家族中活性最高的P450（来自巨大芽孢杆菌的P450 BM 3）来实现这一目的，并通过合理诱变和定向进化的组合来进化BM 3以催化特定功能，利用BBSRC资助的机器人设施进行筛选。开发BM 3的主要原因是a）它是P450与其P450还原酶的天然可溶性融合物，其催化活性通常是多组分人酶的约1000倍，和B）它已经被证明（通过我们自己的实验室和其他地方的工作）这种酶的活性部位是柔韧的，可以被设计成结合和氧化与其“天然”脂肪酸底物完全不同的分子（例如芳族化合物和多环芳族化合物、短链烯烃和烷烃）。学生将结合理性诱变和定向进化（具有合理的靶点，包括Phe 87、Val 78、Ala 264和Leu 181，我们已经证明其对底物选择性的控制至关重要），以使BM 3的反应性多样化，使得其将氧化上述人P450的原型底物：睾酮（3A 4）、右美沙芬（2D 6）、双氯芬酸（2C 9）、地西泮（2C 19）和非那西丁（1A 2）。我们的工作已经表明，BM 3氧化睾酮（但不是在野生型BM 3的正确碳），并且所有其他靶向的人类P450底物具有与各种BM 3突变体氧化的其他化合物相似的分子尺寸和化学特征。我们将使用机器人技术和E.表达BM 3突变体的大肠杆菌细胞提取物，和氧平板技术，其中测量染料连接的氧消耗。增强对关键底物的活性的点突变体将与已知控制底物选择性的区域（I螺旋、F/G和B/C环区域）中随机进化的变体和通过进一步筛选鉴定的改进突变体组合。有机产品将通过GCMS/HPLC方法进行表征，并对优化的酶进行纯化和酶促和结构表征（无配体和底物结合形式）。学生将得到一个并行P450进化项目的PDRA的支持，并将开发新的催化剂，用于产生重要的分子，作为试剂和诊断剂用于商业开发。