Assembly of Artificial Metalloenzymes for Biocatalysis

用于生物催化的人工金属酶的组装

• 批准号: 2741774
• 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: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2741774
• 关键词: Assembly; Artificial; Metalloenzymes; Biocatalysis

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.

生物催化目前正在从利用天然酶到开发具有新反应活性的人造酶方面取得进展。由于天然酶中金属辅因子的范围有限，将互补的有机金属催化剂结合到蛋白质支架中来合成人工金属酶(ARM)是非常有兴趣的。蛋白质支架提供了一个手性环境，提高了催化剂的(立体)选择性，使催化反应能够在水溶液中、在生物相容的条件下进行，并作为生化反应级联的一部分。本项目的目的是制备和组装手臂，例如人工亚胺或酮还原酶，并优化它们的催化活性和对映体选择性。然后，有希望的手臂将通过利用铁载体介导的活跃的细菌铁吸收途径被困在大肠杆菌细胞中，铁载体是由细菌自然产生的分子。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胺或醇。在我们以前工作的基础上，将使用化学共轭技术，如酰胺偶联反应，将主要由d6低自旋金属离子组成的动力学惰性络合物连接到铁载体L偶氮胆碱及其合成类似物的骨架上。一旦纯化和表征，所选蛋白支架，特别是铁载体结合蛋白的结合物的亲和力将被确定。为了指导结构修改，我们将使用前景看好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突变来改变蛋白质提供的环境，以提高催化剂的对映选择性。使用手臂进行催化活性测试将根据我们实验室已经建立的程序进行。产品的形成将通过手性高效液相分析和UV/Vis光谱进行监测。通过电感耦合等离子体发射光谱仪对渗透冲击萃取物进行金属分析，可以确定周质催化剂的浓度。可以改变以优化催化性能的参数包括蛋白质表达水平、组分结构和浓度、孵育时间和pH。