Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Biochemistry and Cellular and Molecular Biology

Major Professor

Engin H. Serpersu

Committee Members

Cynthia Peterson, Dan Roberts, Gladys Alexandre, Jaan Mannik


The aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that detoxifies antibiotics by nucleotidylating at the C4'-OH site. Two thermostable variants T130K and D80Y generated by direct evolution in laboratory differ by only a single residue replacement compared to the wild type mesophilic enzyme. Both variants display enhanced melting temperatures and execute catalysis at temperatures the wild type would be inactive. However, T130K variant still keeps molecular properties of mesophilic enzyme. T130àK130 does not trigger significant change in enzyme’s local flexibility or thermodynamics of ligand binding while D80Y variant has distinct properties in ligand recognition and dynamics. We hypothesize that T130K and D80Y variants adopt different strategies to achieve thermal stability. In this respect, T130K is a heat stable mesophilic enzyme with simply higher melting temperature due to more stabilizing intramolecular interactions and may not be a true “thermophilic” enzyme. Thermophilic variant D80Y, on the other hand, displays higher atomic fluctuations than mesophilic enzyme thus increasing the entropic change associated with enzyme denaturation. Here we attempt to draw a line separating heat resistant enzymes like T130K from true thermophilic enzyme like D80Y.

Numerous studies compared the differences in various structural features of thermophilic/thermostable-mesophilic enzymes in order to reach unifying and general mechanisms of greater thermostability/thermophilicity for such enzymes. To date, not a single molecular feature emerged as the parameter defining “thermophilic” properties. We believe that this is because these comparisons included all heat stable enzymes, some of which may simply be heat stable versions of mesophilic enzymes, such as those with added stabilizing interactions (disulfide bonds or salt bridges) based on structural analyses. In this work, we demonstrated that thermodynamic properties of protein-ligand interactions may yield molecular properties of true thermophilic enzymes by using two heat stable variants of ANT to demonstrate that one of them, T130K is simply a heat stable enzyme with proper ties of the wild type while the other, D80Y, shows properties that are significantly different and alters the dynamics of the enzyme.

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