Engineering a novel chemo-enzymatic cofactor regeneration system
Lead Research Organisation:
University of Nottingham
Department Name: Faculty of Engineering
Abstract
Artificial metalloenzymes are built by chemical modification of proteins with catalytic functionalities that are not available in nature, thus expanding the catalytic scope of enzymes towards non-natural activities. Their active site consists of a hydrophobic pocket, which is large enough to accommodate a chemical catalyst and a non-natural substrate. Precise localisation of the metal complex is difficult to achieve, and few structures of artificial metalloenzymes exist. Therefore, the design of these hybrid catalysts is typically time and resource-consuming work based on intuition and screening. Few examples of computational design have been reported. This project will combine the power of computational and experimental strategies in order to improve the design of hybrid catalysts. In order to fully benefit from the architecture of an enzymatic active site, NAD(P)H-dependent alcohol dehydrogenases (ADHs) will be used as scaffolds. Recycling of the cofactor is very important in biocatalysis. Current enzymatic methods for NADPH recycling lack stability or generate waste co-products, whilst chemical methods are limited by catalyst inactivation at the surface of ADH. The project will investigate the incorporation of chemical catalysts active towards cofactor recycling into the active site of a thermostable alcohol dehydrogenases (TbADH). The catalyst will be positioned in the proximity of the active site by the covalent modification of ADHs at a uniquely reactive cysteine. We will attempt to crystallise the resulting constructs, based on previous reports suggesting crystallisation conditions for this protein. The location of the metal complexes inside the protein will be evaluated either from the crystal structure, or computationally. These structural / computational models will identify useful modifications of the enzyme (genetic mutations) or the ligands (chemical modifications) in order to optimize ligand binding and positioning. The models will be validated by synthesis and testing of the chemical catalysts in the lab.
Organisations
| Description | We incorporated catalysts active towards nicotinamide cofactor recycling at various positions within the hydrophobic pocket of the alcohol dehydrogenase TbADH. We then assessed the catalytic performance of the resulting artificial metalloenzyme variants in the reduction of natural cofactors NAD(P)H, and the hydrophobic mimic 1-benzylnicotinamide. Upon further kinetic characterisation of a promising variant, we observed Michaelis-Menten behaviour with the NAD(P)H cofactors. In contrast, this typical enzyme behaviour was not observed in the reduction of the cofactor mimic. Computational docking data provided insight into these observations, suggesting that the NAD(P)H cofactors bind at a single external site while the cofactor mimic can bind both inside and outside the hydrophobic pocket of TbADH. These results provide a better understanding of interactions between the metal complex, enzyme scaffold and substrate components of artificial metalloenzymes based on TbADH. |
| Exploitation Route | This work provides valuable information in the design of artificial metalloenzymes based on TbADH for the reduction of hydrophobic substrates. |
| Sectors | Chemicals,Pharmaceuticals and Medical Biotechnology |