The Aminoglycoside Nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that detoxifies antibiotics by nucleotidylating at the C4'- OH site. There are several thermostable variants for this enzyme that show a single and two amino acid change in their primary amino acid sequences. It is not clear how one or two residue replacements, which are away from active site and monomer-monomer interface, result in various degrees of changes on enzyme's thermostability. In this work, sedimentation velocity analysis of wild type (mesophilic) and thermostable variants of ANT by analytical ultracentrifugation showed that all the tested enzymes exist as mixtures of monomers and dimers in solution, however, the dissociation constant of dimers steadily increased from the hyperthermophilic to mesophilic variant by ~70-fold. Furthermore, isothermal titration calorimetry studies showed that, at low ionic strength, the dimerization is an endothermic process for wild type (mesophilic) enzyme, while it is an exothermic reaction for all thermophilic variants, where the hyperthermophilic variant shows the largest exothermic enthalpy of dimerization. Dimerization of all enzymes at higher salt concentration (100mM NaCl) became exothermic, again steadily becoming more exothermic with increasing thermophilicity of the variants.

Contribution of subunit-subunit interactions to enhanced thermostability of aminoglycoside nucleotidyltransferase (4') and its variants

248th National Meeting (2014)

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