Thermodynamic characterization of a thermostable antibiotic resistance enzyme, the aminoglycoside nucleotidyltransferase (4′)
The aminoglycoside nucleotidyltransferase (4′) (ANT) is an aminoglycoside-modifying enzyme that detoxifies antibiotics by nucleotidylating at the C4′-OH site. Previous crystallographic studies show that the enzyme is a homodimer and each subunit binds one kanamycin and one Mg-AMPCPP, where the transfer of the nucleotidyl group occurs between the substrates bound to different subunits. In this work, sedimentation velocity analysis of ANT by analytical ultracentrifugation showed the enzyme exists as a mixture of a monomer and a dimer in solution and that dimer formation is driven by hydrophobic interactions between the subunits. The binding of aminoglycosides shifts the equilibrium toward dimer formation, while the binding of the cosubstrate, Mg-ATP, has no effect on the monomer-dimer equilibrium. Surprisingly, binding of several divalent cations, including Mg2+, Mn2+, and Ca2+, to the enzyme also shifted the equilibrium in favor of dimer formation. Binding studies, performed by electron paramagnetic resonance spectroscopy, showed that divalent cations bind to the aminoglycoside binding site in the absence of substrates with a stoichiometry of 2:1. Energetic aspects of binding of all aminoglycosides to ANT were determined by isothermal titration calorimetry to be enthalpically favored and entropically disfavored with an overall favorable Gibbs energy. Aminoglycosides in the neomycin class each bind to the enzyme with significantly different enthalpic and entropic contributions, while those of the kanamycin class bind with similar thermodynamic parameters. © 2012 American Chemical Society.
Publication Source (Journal or Book title)
Jing, X., Wright, E., Bible, A., Peterson, C., Alexandre, G., Bruce, B., & Serpersu, E. (2012). Thermodynamic characterization of a thermostable antibiotic resistance enzyme, the aminoglycoside nucleotidyltransferase (4′). Biochemistry, 51 (45), 9147-9155. https://doi.org/10.1021/bi301126g