Assembly of Artificial Metalloenzymes for Biocatalysis

生物催化正从利用天然酶发展到开发具有新活性的人工酶。由于天然酶中金属辅助因子的范围有限，因此在蛋白质支架中加入互补的有机金属催化剂来制造人工金属酶（arm）是人们非常感兴趣的。蛋白质支架提供了一个手性环境，增加了催化剂的（立体）选择性，使催化反应能够在水溶液中进行，在生物相容性条件下，作为生化反应级联的一部分。该项目的目的是制备和组装arm，例如人工亚胺或酮还原酶，并优化其催化活性和对映体选择性。有希望的arm将被困在大肠杆菌细胞中，利用由铁载体（细菌自然产生的分子）介导的活跃的细菌铁摄取途径。通过这种方式，细菌细胞可以被利用来支持非生物反应，例如生产有价值的对映纯胺或醇。化学偶联技术，如酰胺偶联反应，将被用于将主要由d6低自旋金属离子组成的动力学惰性配合物连接到铁载体l -偶氮chelin和合成类似物的主链上，以我们之前的工作为基础。一旦纯化和表征，将确定缀合物对选定蛋白质支架的亲和力，特别是铁载体结合蛋白。为了指导结构修饰，我们将使用有前途的铁载体锚定催化剂和蛋白质支架进行结晶筛选。获得的晶体结构将表明如何通过诱变来修饰蛋白质所提供的环境，以增加催化剂的对映体选择性。ArMs的催化活性测试将根据我们实验室已经建立的程序进行。产品形成将通过手性高效液相色谱分析和紫外/可见光谱进行监测。采用ICP-OES对渗透冲击萃取物进行金属分析，测定周质催化剂浓度。可以改变的参数，以优化催化性能包括蛋白质表达水平，组分结构和浓度，孵育时间和pH。

Biocatalysis is currently progressing from utilising natural enzymes to the development of artificial enzymes with new reactivities. Since the range of metal cofactors in natural enzymes is limited, it is of great interest to incorporate complementary organometallic catalysts within protein scaffolds to create artificial metalloenzymes (ArMs). The protein scaffold provides a chiral environment that increases the (stereo)selectivity of the catalyst and enables catalytic reactions to be performed in aqueous solution, under biocompatible conditions and as part of biochemical reaction cascades.The aims of this project are to prepare and assemble ArMs, for example artificial imine or ketone reductases, and to optimise their catalytic activity and enantioselectivity. Promising ArMs will then be trapped in E. coli cells by taking advantage of active bacterial iron-uptake pathways that are mediated by siderophores, molecules that are naturally produced by bacteria. In this way, the bacterial cell may be exploited to support abiotic reactions, for example the production of valuable enantiopure amines or alcohols. Chemical conjugation techniques, such as amide coupling reactions, will be used to attach kinetically-inert complexes of mainly d6 low-spin metal ions to the backbone of the siderophore L-azotochelin and synthetic analogues, building upon our previous work. Once purified and characterised, the affinity of the conjugates for selected protein scaffolds, in particular siderophore-binding proteins, will be determined. To guide structural modifications, we will carry out crystallisation screens with promising siderophore-anchored catalysts and protein scaffolds. The crystal structures obtained will indicate how the environment provided by the protein could be modified by mutagenesis to increase the enantioselectivity of the catalysts.Catalytic activity tests with the ArMs will be carried out according to procedures already established in our labs. Product formation will be monitored by chiral HPLC analysis and UV/vis spectroscopy. Periplasmic catalyst concentrations will be determined through metal analysis of osmotic shock extracts by ICP-OES. Parameters that can be varied to optimise catalytic performance include protein expression levels, component structures and concentrations, incubation times and pH.