Molecular determinants of affinity for aminoglycoside binding to the aminoglycoside nucleotidyltransferase(2'')-Ia.
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.
Wright
Edward
E
Department of Biochemistry and Cellular and Molecular Biology, Center of Excellence for Structural Biology, University of Tennessee-Knoxville, Knoxville, Tennessee 37996, USA.
Serpersu
Engin H
EH
D000617:Aminoglycosides / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D016905:Gram-Negative Bacterial Infections / Q000188:drug therapy; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D000617:Aminoglycosides; D001426:Bacterial Proteins; D014867:Water; D009713:Nucleotidyltransferases; C020565:gentamicin 2''-nucleotidyltransferase