Evolution of efficient p450 biocatalysts for human drug metabolite production

人细胞色素P450(P450)是药物/外源物质进入体内代谢过程中的重要酶，也是产生大量类固醇和脂质介质的关键酶。P450催化其底物的氧化代谢，通常具有精致的氧化区域和立体选择性。P450作为化学合成的中间体或具有特定生物功能的高价值脂类介体(如花生四烯酸的衍生物)，在生产含氧官能化分子方面受到了极大的关注，而这些分子往往是非常困难的、低效的和非成本效益的有机合成。在人类P450领域具有特殊价值的将是一组P450产生的关键代谢物的现成来源，这些P450已知对处方药和药物的新陈代谢最重要(特别是CYP3A43、CYP2D6、CYP2C9、CYP2C19和CYP1A2，它们共同氧化和代谢90%的给药药物)。这些代谢物将在人类P450功能的诊断中具有重要价值(即作为人类P450和肝脏代谢的标准)，并作为试剂，例如将有助于确定变异/多态的P450活性(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P450及其双黄素P450还原酶伴侣是完整的膜蛋白，不稳定，活性低。因此，相关产品的产量非常小，利用这些酶的商业过程在财务上是不可行的。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 BM3)，并通过合理突变和定向进化的组合来进化BM3以催化特定功能，利用BBSRC基金已经提供的机器人设施进行筛选。利用BM3的主要原因是：a)它是P450和其P450还原酶的天然和可溶的融合，其催化活性通常是多组分人类酶的约1000倍；b)已经通过我们自己的实验室和其他地方的工作证明，该酶的活性部位是柔韧的，并且可以经过改造来结合和氧化与其“天然”脂肪酸底物(如芳烃和多环芳烃、短链烯烃和烷烃)完全不同的分子。学生将进行理性突变和定向进化的组合(有理性的靶点包括Phe87、Val78、Ala264和Leu181，我们已经证明它们对底物选择性的控制至关重要)，以使BM3的反应活性多样化，以便它将氧化上述人类P450的原型底物：睾酮(3A4)、右美沙芬(2D6)、双氯芬酸(2C9)、地西潘(2C19)和非那西丁(1A2)。我们的工作已经表明，BM3氧化睾酮(但不是在野生型BM3的正确碳处)，而所有其他人类P450底物的分子尺寸和化学特征与各种BM3突变体氧化的其他化合物相似。我们将使用机器人技术和表达BM3突变体的大肠杆菌细胞提取物，以及测量染料连接氧耗的氧板技术来筛选新底物的活性。增强关键底物活性的点突变将与在已知控制底物选择性的区域(在I螺旋、F/G和B/C环区)中随机进化的变体和通过进一步筛选鉴定的改进突变相结合。有机产品将通过GCMS/HPLC法进行表征，并对优化后的酶进行纯化和结构鉴定(以无配体和底物结合的形式)。学生将得到一个并行P450进化项目的PDRA的支持，并将开发用于产生重要分子的新型催化剂，作为商业开发的试剂和诊断。

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.