Thermodynamics Of Aminoglycoside-Enzyme Complexes Yields Clues On Distinguishing Thermophilic Versus Thermostable Variants Of The Amino Glycoside Nucleotidyltransferase 4 ' (Ant4)

The aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that detoxifies many aminoglycoside antibiotics by nucleotidylating them at the C4'-OH site. Two single residue variants with increased thermostability (T130K, D80Y) showed contrasting thermodynamic properties. While T130K shows identical properties to the mesophilic WT, D80Y has stark differences. Data, acquired in H2O and D2O by ITC, demonstrated that solvent reorganization upon ligand binding and changes in low frequency (soft) vibrational modes of the protein are the major contributors for the formation of enzyme-aminoglycoside complexes for WT and T130K. Temperature dependence of ΔCp with D80Y is another difference separating this variant from T130K and WT both of which have a constant ΔCp in the temperature range studied. X-ray structures of these enzymes provide a simple explanation for the thermostability of T130K where the side chain of K130 makes a H-bond with the backbone Oxygen of S90 and brings the unstructured segment around S90 closer to the helical part of K130 and acts like a lynchpin to provide increased thermostability without altering the dynamics and thermodynamics of the protein. The same distance between these two residues are too long for the formation of a H-bond in WT and D80Y. Overall, the data shown in this work suggest that global thermodynamics of ligand-protein interactions and solvent effects may be among the molecular parameters that define thermophilic properties for ANT. Applicability of these findings to other systems remains to be seen.