The development of artificial imine reductases

人工亚胺还原酶的研制

• 批准号: 2106716
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2106716
• 关键词: development; artificial; imine; reductases

The project aims at the development of artificial metalloenzymes that consist of a synthetic organometallic catalyst that is attached via an anchor group to a protein scaffold. By combining the reactivity of an organometallic imine reduction catalyst with the selectivity and biocompatibility of a periplasmic binding protein (PBP), the proposed artificial metalloenzymes aim to catalyse the reduction of imines under mild conditions and in biologically compatible environments. The approach is very versatile since the reactivity of the catalytic centre can be tuned through modifying the synthetic ligand whilst the chiral environment provided by the protein can be modified by genetic means. This project exploits the high-affinity binding of ferric siderophores to their cognate PBPs in the development of novel artificial imine reductases. Individual objectives are to:1) explore the potential of bacterial PBPs as protein scaffolds for the capture of synthetic siderophore-anchored transfer-hydrogenation catalysts2) test the catalytic activity of the artificial enzymes obtained in imine reductions3) attempt the immobilisation of the artificial imine reductases on solid supports4) explore the potential of the artificial imine reductases for the production of synthetic molecules within the periplasm of Gram-negative bacterial cellsMethodology: Chemical conjugation techniques, such as amide coupling or click chemistry will be used to attach kinetically inert piano stool complexes of mainly d6 low-spin metal ions to the backbone of siderophores, such as azotochelin. Once purified and characterised, the conjugates obtained will be incubated with Fe(III) and after equilibration, their affinity for PBPs will be determined to establish how the catalyst-attachment affects the protein-siderophore affinity. To guide structural modifications, we will carry out co-crystallisation screens with promising siderophore-tagged catalysts and PBPs. The co-crystal structures obtained will indicate how the environment provided by the PBP could be modified by mutagenesis to increase the enantioselectivity of the catalysts.For artificial enzyme catalysis to run efficiently within the periplasm of a Gram-negative bacterial cell, the protein scaffold has to be compatible with this environment. PBPs are particularly suitable because of their redundancy and stability. We have identified CeuE as suitable protein scaffold and expressed the protein periplasmically in E. coli, ready for the capture of incoming siderophores-catalyst conjugates. Product formation will be monitored by chiral HPLC analysis of cell supernatant extracts. Periplasmic catalyst concentrations will be determined through metal analysis of osmotic shock extracts by ICP-MS.

该项目旨在开发人工金属酶，该酶由通过锚基团连接到蛋白质支架上的合成有机金属催化剂组成。通过将有机金属亚胺还原催化剂的反应性与周质结合蛋白（PBP）的选择性和生物相容性相结合，所提出的人工金属酶旨在在温和条件下和生物相容的环境中催化亚胺的还原。该方法是非常通用的，因为催化中心的反应性可以通过修饰合成配体来调节，而蛋白质提供的手性环境可以通过遗传手段来修饰。该项目利用铁载体与其同源PBP的高亲和力结合来开发新型人工亚胺还原酶。个别目标是：1）探索细菌PBP作为捕获合成铁载体锚定的转移-氢化催化剂的蛋白质支架的潜力2）测试在亚胺还原中获得的人工酶的催化活性3）尝试将人工亚胺还原酶固定在固体支持物上4）探索人工亚胺还原酶用于在革兰氏阴性细菌细胞周质内产生合成分子的潜力。化学结合技术，例如酰胺偶联或点击化学将用于将主要是D 6低自旋金属离子的动力学惰性的平粪便络合物连接到铁载体的主链上，例如偶氮螯铁蛋白。一旦纯化和表征，将获得的缀合物与Fe（III）孵育，并且在平衡后，将测定它们对PBP的亲和力以确定催化剂附着如何影响蛋白质-铁载体亲和力。为了指导结构修饰，我们将用有前途的铁载体标记的催化剂和PBP进行共结晶筛选。共晶结构的获得将表明PBP提供的环境如何通过诱变来改变以增加催化剂的对映体选择性。为了使人工酶催化在革兰氏阴性菌细胞的周质内有效地运行，蛋白质支架必须与该环境相容。PBPs由于其冗余性和稳定性而特别适合。我们已经确定CeuE作为合适的蛋白支架，并在E.大肠杆菌，准备捕获传入的铁载体-催化剂缀合物。将通过对细胞上清液提取物进行手性HPLC分析来监测产物形成。通过ICP-MS对渗透压休克浸提液进行金属分析，测定周质催化剂浓度。