Padley, Henry
(2024)
Structure-guided design of artificial metalloenzymes based on alcohol dehydrogenase for transfer hydrogenation.
PhD thesis, University of Nottingham.
Abstract
Artificial metalloenzymes (ArMs) combine the advantages of enzyme and synthetic catalysts to offer novel solutions to the synthesis of high-value fine chemicals. ArMs are assembled by incorporation of an organometallic catalyst into a protein scaffold. This enables the exertion of control over the chemical environment of the catalyst via genetic manipulation of the scaffold, providing one of the key advantages of ArMs over small-molecule catalysts. However, rational genetic optimisation requires an understanding of the interactions between the protein, catalyst, and substrate within the ArM. The incompleteness of such knowledge has been highlighted as a relative gap for exploitation within the wider ArM field. In particular, there is little structural information available on ArMs based on enzymatic scaffolds. These systems offer a promising alternative to highly successful ArMs based on non-enzymatic scaffolds. The naturally evolved architecture of the enzyme can be used to the advantage of ArM catalyst or substrate binding.
Here, we report kinetic and structural insights into ArMs based on an alcohol dehydrogenase (ADH) scaffold for the reduction of nicotinamide cofactors, followed by attempts to expand ArM functionality towards other transfer hydrogenation reactions. The ArM system, which was established and developed by other members of the research group, is based upon covalent anchoring of rhodium piano-stool complexes to T. brockii ADH. A section of the previous work began to explore different anchoring locations for the catalyst within the TbADH scaffold, towards optimisation of ArM catalytic performance on different nicotinamide substrates. However, full kinetic characterisation of these ArM variants, and detailed structural information useful for rational optimisation efforts was lacking.
In the present work, rhodium-TbADH ArM variants based on two of the previous covalent anchoring locations (TbADH residue locations 37 and 243), were subject to full kinetic characterisation and docking studies. Location 37 was found to be favourable with regards to ArM affinity for natural NAD(P)+ cofactors, which was estimated using values of the Michaelis constant KM. This could be explained by reduced obstruction of the entrance to the TbADH nicotinamide cofactor binding pocket. These results prompted the design of a new variant based on anchoring of the catalyst to location 110 which was subject to the same docking and kinetics analyses. As hypothesised, the results indicate further improvement in ArM affinity for the NAD(P)+ substrates. In particular, a greater overlap of the NADP+ binding site with the wildtype TbADH binding site of this cofactor was predicted by docking. This suggests an improved utilisation of the naturally evolved TbADH nicotinamide binding pocket.
The X-ray crystal structure of a residue location 110-modified TbADH ArM co-crystallised with NADP+ also indicates a near-wildtype binding site of the nicotinamide cofactor. While this structure also shows the covalently bound catalyst in an alternative non-catalytic orientation, it appears possible that flexibility of the catalyst in solution allows movement into the catalytically active orientation, which was predicted by docking.
Additionally, the same docking and kinetics studies were completed with the smaller nicotinamide mimic BNA+. All rhodium-TbADH ArM variants displayed a lower affinity for this mimic in comparison to the natural NAD(P)+ cofactors, predominantly owing to comparatively fewer favourable protein-ligand interactions.
Finally, preliminary experiments were completed to explore the functionality of iridium-TbADH ArMs for nicotinamide and other transfer hydrogenation applications. Very low levels of nicotinamide reduction activity of ArMs modified at residue locations 37 and 110 provide proof of principle for the functionality of these ArMs.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Pordea, Anca |
| Keywords: |
Artificial metalloenzymes; Alcohol dehydrogenase scaffold; Transfer hydrogenation reactions |
| Subjects: |
T Technology > TP Chemical technology |
| Faculties/Schools: |
UK Campuses > Faculty of Engineering > Department of Chemical and Environmental Engineering |
| Item ID: |
77857 |
| Depositing User: |
Padley, Henry
|
| Date Deposited: |
18 Jul 2024 04:40 |
| Last Modified: |
18 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/77857 |
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