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

“人性化”一种催化高效的细菌加氧酶，用于高价值代谢物的生产

• 批准号: 1788713
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1788713
• 关键词: Humanizing; catalytically; efficient; bacterial; oxygenase

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.

治疗药物在体内广泛代谢，主要参与的酶是细胞色素P450（P450）。P450识别外来分子并催化氧插入反应，使它们更具极性/可溶性，并靶向它们进行进一步的酶促修饰和排泄。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抗肿瘤活性。FDA和其他监管机构要求对主要代谢物和母体药物进行检测。然而，由于通过合成化学以区域/立体选择性方式插入氧原子涉及的困难，并且由于人P450通常是缓慢/不稳定的膜结合酶，其需要另一种膜结合还原酶来发挥功能，因此这种P450代谢物的产生具有挑战性。制备大量有价值的药物代谢物的替代途径涉及工程化更有效的P450来识别/氧化这些药物。这种酶是细菌P450 BM 3，一种可溶性P450天然融合到其还原酶，并具有最高的P450催化速率（比人类P450快约1000倍）。已经产生了氧化包括咖啡因和奥美拉唑在内的药物的BM 3突变体。该项目将涉及进一步改进现有的变体和新的BM 3突变体的工程设计，以将底物范围扩大到镇痛剂，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进行，涉及的研究包括蛋白质工程、P450-药物复合物的结构生物学和形成的药物代谢物分析（使用HPLC和GC-MS方法）。该项目是与Cypex Ltd的工业合作，Cypex Ltd在该领域拥有广泛的专业知识。