Molecular Determinants of Affinity for Aminoglycoside Binding to the Aminoglycoside Nucleotidyltransferase(2‘ ‘)-Ia

Abstract

One of the most commonly occurring aminoglycoside resistance enzymes is aminoglycoside 2''-O-nucleotidyltransferase [ANT(2'')]. In the present study molecular determinants of affinity and specificity for aminoglycoside binding to this enzyme are investigated using isothermal titration calorimetry (ITC). Binding of aminoglycosides is enthalpically driven accompanied by negative entropy changes. The presence of metal-nucleotide increases the affinity for all but one of the aminoglycosides studied but has no effect on specificity. The substituents at positions 1, 2', and 6' are important determinants of substrate specificity. An amino group at these positions leads to greater affinity. No correlation is observed between the change in affinity and enthalpy. At the 2' position greater affinity results from a more negative enthalpy for an aminoglycoside containing an amino rather than a hydroxyl at that position. At the 6' position the greater affinity for an aminoglycoside containing an amino substituent results from a less disfavorable entropic contribution. The thermodynamic basis for the change in affinity at position 1 could not be determined because of the weak binding of one of the aminoglycoside substrates, amikacin. The effect of increasing osmotic stress on affinity was used to determine that a net release of approximately four water molecules occurs when tobramycin binds to ANT(2''). No measurable net change in the number of bound water molecules is observed when neomycin binds the enzyme. Data acquired in this work provide the rationale for the ability of ANT(2'') to confer resistance against kanamycins but not neomycins.

Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase‐VIa

“…The net deprotonation, Δ n, was −0.22 ± 0.1 and −0.32 ± 0.1 for the binary and ternary complexes, respectively. This is quite different than what was observed with highly promiscuous aminoglycoside modifying enzymes where all titrations yielded positive slopes in such plots indicating an overall net proton uptake …”

“…The last thermodynamic parameter, the change in heat capacity (ΔCp), was determined from the temperature dependence of the binding enthalpy. Solvent reorganization is one of the main contributors to ΔCp, and earlier observations with several, highly promiscuous, aminoglycoside modifying enzymes showed that solvent reorganization played an important role in the formation of enzyme–aminoglycoside complexes . Therefore, to determine effects of solvent, binding experiments were performed in H 2 O and D 2 O.…”

“…Thermodynamic parameters of enzyme–ligand complexes of aminoglycoside N3 ‐acetyltransferases are known only for the highly promiscuous enzymes, the aminoglycoside N3‐ acetyltransferase‐IIIb (AAC‐IIIb), and moderately promiscuous, the aminoglycoside N3 ‐acetyltransferase‐IIa (AAC‐IIa) . Thermodynamic data on several other, highly promiscuous, aminoglycoside modifying enzymes that catalyze phosphorylation and nucleotidylation are also available . However, there is no thermodynamic data available for an aminoglycoside modifying enzyme with highly limited substrate profile.…”

Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic, structural and kinetic study

“…By isothermal titration calorimetry (ITC), we determined the thermodynamic parameters, and consequently the binding affinities, of aminoglycoside binding to APH(2″)-IVa. Although ITC has been used extensively to characterize aminoglycoside binding to APH(3′)-IIIa [29] , [30] , [31] , [32] or to other aminoglycoside modifying enzymes [33] , [34] , [35] , [36] , [37] , [38] , [39] , this work brings the first data for APH(2″)-IVa. Fig.…”

“…This is similar to what is observed with the aminoglycoside nucleotidyltransferase(2″)-Ia, to which neomycins bind tighter but are not substrates of the enzyme. In this case, the 2″-site becomes too distant to the α-phosphate group for a direct nucleophilic attack [35] . Thus, unfavorable orientation of the 2″-site in the more constrained binding site on APH(2″)-IVa may be correlated with its inability to catalyze phosphotransfer with 4,5-disubstituted aminoglycosides.…”