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Biochim Biophys Acta. 2016 Apr;1860(4):802-13. doi: 10.1016/j.bbagen.2016.01.016. Epub 2016 Jan 21.

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

Author information

1
CNRS, FRE3689 - Université de Montpellier, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, F-34293 Montpellier, France.
2
CNRS, UMR5048 - Université de Montpellier, Centre de Biochimie Structurale, F-34090 Montpellier, France; INSERM, U1054, F-34090 Montpellier, France.
3
Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, UK.
4
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA.
5
CNRS, FRE3689 - Université de Montpellier, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, F-34293 Montpellier, France. Electronic address: corinne.lionne@cpbs.cnrs.fr.

Abstract

BACKGROUND:

Aminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step.

METHODS:

We used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements.

RESULTS:

For the first time, Kd values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio Kd/Km, there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction.

CONCLUSIONS:

APH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2' and 6', but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3')-IIIa, an intermediate containing product is preponderant during the steady state.

GENERAL SIGNIFICANCE:

This intermediate may constitute a good target for future drug design.

KEYWORDS:

Aminoglycoside modifying enzymes; Electrostatic potential calculations; Isothermal titration calorimetry; Molecular dynamics simulations; Transient kinetics; X-ray crystallography

PMID:
26802312
PMCID:
PMC4769084
DOI:
10.1016/j.bbagen.2016.01.016
[Indexed for MEDLINE]
Free PMC Article

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