"Humanizing" a catalytically efficient bacterial oxygenase for high high value metabolite production

治疗药物在体内广泛代谢，主要涉及的酶是细胞色素P450(P450)。P450识别外源分子，并催化氧插入反应，使它们更具极性/更易溶，并针对它们进行进一步的酶修饰和排泄。越来越多的人认识到，由P450介导的药物氧化形成的代谢物可能具有药理活性。FDA和其他监管机构要求对主要代谢物和母药进行测试。然而，由于合成化学中以区域/立体选择性的方式插入氧原子存在困难，而且人类P450通常是缓慢/不稳定的膜结合酶，需要另一种膜结合还原酶才能发挥作用，因此生产这样的P450代谢物是具有挑战性的。制造大量有价值的药物代谢物的另一种方法是设计一种更有效的P450来识别/氧化这些药物。这种酶就是细菌P450 BM3，一种天然与其还原酶融合的可溶性P450，具有最高的P450催化速率(约为人类P450的1000倍)。已经产生了氧化咖啡因和奥美拉唑等药物的BM3突变体。该项目将包括进一步改进现有的变种和设计新的BM3突变体，以将底物范围扩大到止痛药、类固醇和其他重要化合物。该项目将在曼彻斯特生物技术研究所完成，涉及的研究包括蛋白质工程、P450-药物复合体的结构生物学以及形成的药物代谢物的分析(使用高效液相色谱和GC-MS方法)。该项目是与Cypex有限公司的行业合作，后者在该领域拥有广泛的专业知识。

Therapeutic drugs are extensively metabolised in the body, with the main enzymes involved being cytochromes P450 (P450s). P450s recognize foreign molecules and catalyse oxygen insertion reactions to make them more polar/soluble, and to target them for further enzymatic modification and excretion. There is increasing recognition that metabolites formed by P450-mediated oxidation of drugs may be pharmacologically active. The FDA and other regulatory agencies require testing of major metabolites as well as parent drugs. However, production of such P450 metabolites is challenging due to difficulties involved in inserting oxygen atoms in a regio-/stereo-selective manner by synthetic chemistry, and since human P450s are typically slow/unstable membrane-bound enzymes that require another membrane-bound reductase to function. An alternative route to making substantial amounts of valuable drug metabolites involves engineering a more efficient P450 to recognize/oxidize these drugs. Such an enzyme is the bacterial P450 BM3, a soluble P450 naturally fused to its reductase and possessing the highest P450 catalytic rate (~1000-fold faster than human P450s). BM3 mutants that oxidize drugs including caffeine and omeprazole have been generated. This project will involve further improvement of existing variants and engineering of novel BM3 mutants to expand the substrate range to analgesics, steroids and other important compounds. The project will be done in the Manchester Institute of Biotechnology and will involve research including protein engineering, structural biology of P450-drug complexes and analysis of drug metabolites formed (using HPLC- and GC-MS methods). The project is an industrial collaboration with Cypex Ltd, who have extensive expertise in the area.