Assembly of Artificial Metalloenzymes for Biocatalysis

目前生物催化正从利用天然酶向开发具有新反应活性的人工酶发展。由于天然酶中的金属辅因子的范围是有限的，因此将互补的有机金属催化剂并入蛋白质支架中以产生人工金属酶（ArMs）是非常有趣的。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，并使催化反应能够在水溶液中进行，在生物相容性条件下和作为生化反应级联的一部分。该项目的目的是制备和组装ArMs，例如人工亚胺或酮还原酶，并优化其催化活性和对映选择性。有希望的武器将被困在E。大肠杆菌细胞通过利用由铁载体介导的活性细菌铁吸收途径，铁载体是细菌天然产生的分子。以这种方式，可以利用细菌细胞来支持非生物反应，例如生产有价值的对映体纯胺或醇。化学共轭技术，如酰胺偶联反应，将被用来连接动力学惰性的配合物，主要是d 6低自旋金属离子的铁载体L-azotochelin和合成类似物的骨干，建立在我们以前的工作。一旦纯化和表征，将确定缀合物对所选蛋白质支架，特别是铁载体结合蛋白的亲和力。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改变蛋白质提供的环境，以增加催化剂的对映选择性。将根据我们实验室已经建立的程序进行ArMs的催化活性测试。将通过手性HPLC分析和UV/维斯光谱法监测产物形成。通过ICP-OES对渗透压休克浸提液进行金属分析，测定周质催化剂浓度。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。

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.