Abstract 1634 Distinct structural regions controlling the quaternary structure of a thermophilic esterase

The quaternary structure of an enzyme controls essential aspects of its function, including its enzymatic activity, folding, and stability. In this study, we investigated factors controlling the high quaternary structure stabilization of TM0077, a thermostable acetyl esterase from Thermotoga maritima. Based on its three-dimensional structure, three distinct subregions of TM0077 were hypothesized to control the formation of its quaternary structure and to contribute to its high thermal stability. To investigate this hypothesis, we made significant deletions and substitutions at two of these regions – the N-terminal helix and three helix bundle – resulting in changes to the protein's folding, oligomeric state, catalytic activity, and enzymatic efficiency. Even with these large amino acid deletions, each of the variants were purified and found to be relatively stable with TM values > 60°C, which although lower than the wild-type TM (105°C) is above the average human protein TM (50°C). Notable shifts were also observed in their oligomeric states, with some variants having a larger oligomeric state than the hexameric state of wild-type TM0077. Additional impacts of these substitutions were also determined on the catalytic activity and enzymatic efficiency of TM0077, where relative shifts in enzymatic activity varied widely based on the mutation. Together these diverse thermal stability, oligomeric state, and kinetic activity data indicate a structural connection between the oligomeric state of TM0077 and its biological functionality, notably seen in the N-terminal helix deletion. Following up on the important role of the N-terminus, pinpoint mutations are being constructed within this region to determine which interactions were key to oligomeric state formation in this thermophilic enzyme.