The development of artificial imine reductases

该项目旨在开发人工金属酶，它由合成的有机金属催化剂组成，通过锚基团附着在蛋白质支架上。通过将有机金属亚胺还原催化剂的反应性与外质结合蛋白（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和生物相容环境下催化亚胺还原。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。本项目利用铁铁载体与其同源PBPs的高亲和力结合来开发新型人工亚胺还原酶。个人目标是：1)探索细菌PBPs作为捕获合成铁载体锚定转移氢化催化剂的蛋白质支架的潜力；2)测试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载体上；4)探索人工亚胺还原酶在革兰氏阴性细菌周质内合成分子生产的潜力细胞方法：化学偶联技术，如酰胺偶联或点击化学将被用于将主要由16个低自旋金属离子组成的动力学惰性钢琴凳配合物连接到铁载体（如偶氮螯合蛋白）的主干上。纯化和表征后，得到的偶联物将与Fe（III）孵育，平衡后，将确定它们对PBPs的亲和力，以确定催化剂附着如何影响蛋白质-铁载体的亲和力。为了指导结构修饰，我们将对有前途的铁载体标记催化剂和PBPs进行共结晶筛选。所获得的共晶结构将表明PBP所提供的环境如何通过诱变来提高催化剂的对映选择性。为了使人工酶催化在革兰氏阴性细菌细胞的周质内有效地运行，蛋白质支架必须与这种环境兼容。由于其冗余性和稳定性，PBPs特别适用。我们已经确定了CeuE作为合适的蛋白支架，并在大肠杆菌中表达了该蛋白，准备捕获进入的铁载体-催化剂偶联物。产品形成将通过手性高效液相色谱分析细胞上清提取物监测。采用ICP-MS对渗透休克提取液进行金属分析，测定周质催化剂浓度。

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.