Investigation Of The Molecular Mechanisms Which Result In Aminoglycoside Nucleotidyltransferase 4' (Ant4) Variants With Different Levels Of Thermostability

The aminoglycoside nucleotidyltransferase 4′ (ANT) is a homodimeric enzyme that detoxifies a large number of aminoglycoside antibiotics by nucleotidylating them at the C4′-OH site. Two thermostable variants for this enzyme show only single amino acid changes in their primary amino acid sequences (T130K, D80Y). D80Y has a higher melting temperature compared to T130K. Our previous studies indicate that T130K mimics the WT enzyme in terms of protein dynamics and thermodynamics of enzyme-ligand interactions while the D80Y exhibits different behaviors in both aspects. This led us to hypothesize that the T130K is just a thermostable version of the mesophilic WT exhibiting the same behaviors while the D80Y is the true thermophilic variant with distinct properties. Our recent work shows that the thermodynamic parameters of the binary enzyme-aminoglycoside complexes of these variants show highly significant differences. The data, acquired in H2O and D2O by isothermal titration calorimetry demonstrated that solvent reorganization upon ligand binding show large differences between the two variants. The heat capacity change (ΔCp) also show antibiotic-dependent differences between the two variants. The X-ray structures of both variants show that the aminoglycoside neomycin is positioned differently at the active site of T130K as compared to D80Y, in which the tyrosine substitution alters the geometry of binding site. This structural difference explains why we observe differences in the thermodynamics of the ligand binding properties between these variants. Furthermore, temperature affects the monomer-dimer equilibrium of these enzymes differently. Thus, data shown in this work suggest that dynamic properties of proteins, thermodynamics of ligand-protein interactions, solvent effects and monomer-dimer equilibrium may be among the molecular parameters that separate thermophilic proteins from simply those that are thermostable but otherwise identical to the mesophilic counterparts.