Combining theory and experiment to explain the evolution of antibiotic resistance

Enzyme activity exhibits temperature dependence, manifest as optimum temperature of maximum activity, curvature in Eyring plots and activity loss at both high and low, temperatures. This has generally been ascribed to protein instability; i.e. loss of structure and activity at temperatures outside the normal range. However, recent findings show this to be incorrect: for many enzymes, activity drops significantly above optimum temperature without unfolding. This may be explained by the recently developed Macromolecular Rate Theory, holding that the temperature dependence of enzyme activity is accounted for by the activation heat capacity (i.e. the change in heat capacity between ground state and transition state). This is a significant advance in understanding and explaining enzyme thermoadaptation and evolution. Evidence comes from MMRT fitting of experimental data, and the ability of single point mutations to shift optimum temperature of the alpha-glucosidase MalL. Very recently we have shown that enzyme activation heat capacities can be calculated from molecular dynamics (MD) simulations which can then identify changes in protein dynamics causing this
behaviour. Here we apply these ideas to study enzymes central to antibiotic resistance: beta-lactamases, which hydrolyse beta-lactam antibiotics. Successive generations of beta-lactams, or mechanism-based inhibitors designed to overcome resistance, have engendered enzyme variants with altered or expanded activity spectra. Many mutations in these are remote from the active site, making it difficult to rationalise effects on activity in terms of substrate recognition or catalytic mechanism. We hypothesise that such mutations operate instead by affecting the heat capacities of the Michaelis and/or transition state complexes, and that their optimal values depend upon substrate, as well as enzyme. In this project, the student will investigate the temperature dependence of betalactam hydrolysis by beta-lactamases and natural point variants, with the aim of obtaining activation heat capacities for selected enzyme:substrate combinations.
Experiments will be complemented by simulations (MD over extended time scales together with QM/MM reaction mechanism modelling) based upon experimental crystal structures. Simulations will provide a molecular-level analysis of experimental data, predict mutations for 3 / 16 experimental analysis, identify how these affect protein dynamics and predict activation heat capacities for comparison with experiment. This project will advance fundamental understanding of enzyme catalysis and identify routes by which beta-lactamases acquire activity against new substrates, potentially enabling prediction of resistance emergence to new beta-lactams. The student will acquire expertise in recombinant protein production, enzyme kinetics and structural biology (X-ray crystallography) methods as well as in computational enzymology and molecular modelling.