F420: Characterisation and Catalysis in Mycobacterial Enzymes

Abstract

Experimental UV-visible spectroscopy, computational quantum chemistry and molecular dynamics (MD) simulations have been used to characterise the deazaflavin cofactor F420 and its role in the catalytic activity of a newly classified superfamily of flavin/deazaflavin dependent oxidoreductases (FDORs). Two FDORs were selected to represent the enzyme family as case studies in the detailed investigations; Deazaflavin dependent nitroreductase (Ddn) and Rv2074, which is a novel biliverdin reductase. The main findings of the thesis are as follows. The protonation/deprotonation state of the F420H2 cofactor species was determined in the Ddn active site using experimental UV-visible spectroscopy and computationally simulated spectra. Ddn was found to bind F420H2 in its deprotonated state. This is important as it implies that the subsequent protonation step following the obligate hydride transfer requires a different proton donor for the completion of the reaction. Using the above results and the published crystal structure of Ddn as a starting point, the MD simulations helped reveal the roles the active site residues play in the catalytic mechanism of pretomanid activation. An interesting outcome of this investigation was the role of three non-binding tyrosine residues that formed a hydrophobic barrier, which shielded the hydride transfer site. Hydrophobic shielding of the hydride transfer site by a non-binding tyrosine was also observed in the second case study, Rv2074. As the studies presented in this thesis are the first to use computational modelling on members of the FDOR family, it is unclear whether other members that use F420H2 as a cofactor also demonstrate similar hydrophobic shielding behaviour. Nonetheless, Rv2074 and Ddn belong to different subfamilies in the FDOR superfamily and outside of a couple of conserved residues, they have vastly different active sites with different residues. The conserved residues were found to be involved in binding F420 and hydrophobic shielding. As this feature is found to be conserved in members from different subfamilies, it could provide new insights into the importance of this feature in how F420 dependent reductases catalyse reactions.

Finally, high level ab initio calculations were used to determine the reaction pathway of the pretomanid activation in Ddn, the molecular structures of the transition states and intermediates involved in the reaction pathway and their associated relative energy barriers. The initial hydride transfer step was determined to be rate limiting. After hydride transfer the hydrophobic shield is disrupted allowing exposure of the active site to bulk solvent so proton transfer can occur from a hydroxonium ion. The product then undergoes a series of spontaneous intramolecular reactions to form the final reactive nitrogen species. The detailed characterisation of this mechanism could be utilised in the future development of pro-drugs that would undergo similar activation reactions.