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

Biochimica Et Biophysica Acta
Elise KaplanCorinne Lionne

Abstract

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. 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. 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 obtai...Continue Reading

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Citations

Sep 19, 2016·Biochimica Et Biophysica Acta. General Subjects·Nadia LebanCorinne Lionne
Jan 4, 2018·Chemical Society Reviews·Nishad Thamban Chandrika, Sylvie Garneau-Tsodikova
Dec 4, 2019·Acta Crystallographica. Section D, Structural Biology·Clyde A SmithSergei B Vakulenko
Apr 11, 2018·ACS Infectious Diseases·Georgina CoxGerard D Wright

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Methods Mentioned

BETA
isothermal titration calorimetry
biosensor
gel filtration
phosphotransferase
phosphotransferases
X-ray

Software Mentioned

TLSMD
APBS
PHENIX
NAMD
VMD
AutoBuild
GOLD
Refmac
Origin
PDB2PQR

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Bacterial Protein Structures (ASM)

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