Assembly of Artificial Metalloenzymes for Biocatalysis

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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.