Temperature Sensitive Contact Analysis Directs Hyperactive Enzyme Design.

Homologous mesostable and thermostable enzymes exhibit markedly different thermostabilty and activities as a result of their unique amino acid sequences. This is a necessary consequence of an organism’s metabolism and environmental pressures of which temperature is the most prominent.  The mechanisms by which sequence specific interactions tune the activity of an enzyme to a particular temperature is of fundamental importance for understanding protein functional dynamics and has significant implications for the design and application of synthetic enzymes. To this end we recently designed hybrid thermophilic/mesophilic variants of the C domain of bacterial Enzyme I (EIC) with modulated activity by hybridizing the disordered catalytic loops of each wild type with the remainder of the other wild type’s C domain.  In order to reveal the residue level interactions encoding enzymatic activities, we employed Hamiltonian Replica Exchange Molecular Dynamics (HREMD) to sample the conformational ensemble across temperature of the disordered catalytic loops of these 4 EIC homologs.  Employing Principal Component Analysis (PCA), we extracted and ranked residues according to their contact frequencies’ temperature sensitivity. We hypothesize that these residues have the strongest effect in tuning the enzymes’ activities to their physiological temperatures. To test our hypothesis we assayed 3 mutant thermophile EIC enzymes with mutations based on these most temperature sensitive contacts.  We found a minimal mutation set to the thermophilic homolog (inactive at low temperature) that increased its activity at low temperature substantially toward that of the hybrid homolog.  These results suggest an efficient computational approach for designing hyperactive thermophilic enzymes and a novel analysis method for studying protein dynamics across temperature and identifying key dynamical features not otherwise apparent.