Investigation on the molecular mechanism of 2OG-oxygenases and serine β-lactamases through computational chemistry

This thesis describes studies on the molecular mechanisms of 2OG-oxygenases and serine β-lactamases, and work towards the decomposition of the thermodynamics of solvation processes using Quantum Mechanics (QM), Molecular Dynamics (MD), and Free Energy Perturbation (FEP) methods.
The first chapter gives a brief introduction to quantum chemistry and free energy perturbation methods as applied to the concept of potential energy surface and transition state theory. Chapter 2 describes QM and MD calculations to investigate recyclisation and hydrolysis in avibactam mediated serine β-lactamase inhibition. β-Lactams inhibit penicillin-binding proteins (PBPs) and serine β-lactamases by acylation of a nucleophilic active site serine. Avibactam is approved for clinical use in combination with a cephalosporin, and is a breakthrough non β-lactam inhibitor, which also inhibits via serine acylation. QM and MD calculations on the avibactam-mediated inhibition of a clinically relevant cephalosporinase reveal recyclisation of the avibactam derived carbamoyl complex is favored over hydrolysis. In contrast, analogous recyclization in β- lactam mediated inhibition is disfavored. Avibactam recyclization is promoted by a proton shuttle, a 'structural' water protonating the nucleophilic serine, and stabilization of negative charge on the avibactam derived carbonyl oxygen.


The results reveal the potential of calculations for distinguishing between bifurcating pathways and in generating hypotheses for predicting resistance. The inability of β- lactams to undergo recyclization may be an Achilles heel, but one that might be addressed by reversibly binding inhibitors.
Chapter 3 describes work using DFT calculation on the selectivity of the hydroxylation of ankyrin repeat domain substrates as catalyzed by a 2-oxoglutarate (2OG) dependent oxygenase. Factor inhibiting hypoxia inducible factor (FIH) is a promiscuous protein hydroxylase that typically catalyses hydroxylations at the C-3 position of protein residues. Amongst residues hydroxylated by FIH, leucine has drawn special interest from a stereochemical perspective. Based on ab initio density functional calculations at the B3LYP/def2-TZVP level of theory, structural and energetic features of the FIH catalysed hydroxylation of L- and D-leucine were investigated. The DFT and experimental results show that L- and D-leucine are oxidized to (3R)-β-hydroxy-L-leucine and (3S)-β- hydroxy-D-leucine, respectively. D-, but not L, -leucine undergoes a double hydroxylation to form β,γ-dihydroxy-D-leucine. The results imply that the difference in the catalytic actions of FIH on L- and D-leucine stems from the different complexation modes in the active site caused by a change in C-α stereochemistry. X-ray crystallography analyses provide evidence for the proposed catalytic mechanisms.


Despite the importance of molecular hydration entropy (ΔShyd) in chemical and biological processes, the accurate calculation of ΔShyd is difficult due to the complexities of solute-water interactions. The studies in chapter 4 concerned a novel method for accurately estimating ΔShyd based on a modified thermodynamic decomposition approach. The key feature of the method is that solute-water interactions are decomposed into hydrophobic and electrostatic parts to calculate their respective contributions to ΔShyd. Although free energy perturbation (FEP) methods have been employed widely, the poor convergent behavior of the van der Waals interaction term in the potential function limits its accuracy and robustness. The new method combines the FEP approach and the scaled particle theory (or information theory) to separately calculate the electrostatic solute-water interaction term (ΔSelec) and the hydrophobic contribution (the cavity formation) entropy (ΔScav). The method appears to be effective with a substantial accuracy enhancement in ΔShyd estimation compared to conventional FEP calculations. ΔScav appears to dominate over ΔSelec in magnitude even in the case of polar solutes, implying that the major contribution to the entropic cost for hydration comes from the formation of a solvent-excluded volume. The method thus enhances the accuracy of ΔShyd prediction by complementing the conventional full FEP method.

Advisor: Schofield, C