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

• Published in: Biochimica et biophysica acta Vol. 1860; no. 4; pp. 802 - 813
• Main Authors: Kaplan, Elise, Guichou, Jean-François, Chaloin, Laurent, Kunzelmann, Simone, Leban, Nadia, Serpersu, Engin H., Lionne, Corinne
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2016; Elsevier; Elsevier Pub. Co
• Subjects: Aminoglycoside modifying enzymes; Aminoglycosides - chemistry; Aminoglycosides - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biochemistry, Molecular Biology; Chemical Sciences; Cheminformatics; Electrostatic potential calculations; Enterococcus - enzymology; Isothermal titration calorimetry; Kinetics; Life Sciences; Molecular dynamics simulations; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Protein Binding; Static Electricity; Structural Biology; Transient kinetics; X-ray crystallography; X-ray crystallography; Electrostatic potential calculations; NCNO; Transient kinetics; PMF; LE; Aminoglycoside modifying enzymes; Isothermal titration calorimetry; Molecular dynamics simulations; APH(3′); APH(2″)
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2016.01.016

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 obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction. 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. This intermediate may constitute a good target for future drug design. [Display omitted] •Aminoglycosides with amino groups in 2′ and 6′ are best ligands for APH(2″)-IVa.•Binding of aminoglycosides on APH is mainly driven by electrostatic interactions.•Enzyme affinity seems to anti-correlate with the molecular weight of the ligand.•With different aminoglycosides, the ratio Kd/Km varies, but not transient kinetics.•A product release step is rate-limiting for the overall reaction.