The development of artificial imine reductases
Lead Research Organisation:
University of York
Department Name: Chemistry
Abstract
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 catalysts
2) test the catalytic activity of the artificial enzymes obtained in imine reductions
3) attempt the immobilisation of the artificial imine reductases on solid supports
4) explore the potential of the artificial imine reductases for the production of synthetic molecules within the periplasm of Gram-negative bacterial cells
Methodology: 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.
Individual objectives are to:
1) explore the potential of bacterial PBPs as protein scaffolds for the capture of synthetic siderophore-anchored transfer-hydrogenation catalysts
2) test the catalytic activity of the artificial enzymes obtained in imine reductions
3) attempt the immobilisation of the artificial imine reductases on solid supports
4) explore the potential of the artificial imine reductases for the production of synthetic molecules within the periplasm of Gram-negative bacterial cells
Methodology: 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.
