Thermodynamics of aminoglycoside binding to aminoglycoside-3 '-phosphotransferase IIIa studied by isothermal titration calorimetry

The aminoglycoside-3'-phosphotransferase IIIa [APH(3')-IIIIa] phosphorylates aminoglycoside antibiotics and renders them ineffective against bacteria. APH(3')-IIIa is the most promiscuous aminoglycoside phosphotransferase enzyme, and it modifies more than 10 different aminoglycoside antibiotics. A wealth of information exists about the enzyme; however, thermodynamic properties of enzyme-aminoglycoside complexes are still not known. This study describes the determination of the thermodynamic parameters of the binary enzyme-aminoglycoside and the ternary enzyme-metal-ATP-aminoglycoside complexes of structurally related aminoglycosides using isothermal titration calorimetry. Formation of the binary enzyme-aminoglycoside complexes is enthalpically driven and exhibits a strongly disfavored entropic contribution. Formation of the ternary enzyme - metal-ATP-aminoglycoside complexes yields much smaller negative DeltaH values and more favorable entropic contributions. The presence of metal-ATP generally increases the affinity of aminoglycosides to the enzyme. This is consistent with the kinetic mechanism of the enzyme in which ordered binding of substrates occurs. However, the observed DeltaH values neither correlate with kinetic parameters k(cat), K-m, and k(cat)/K-m nor correlate with the molecular size of the substrates. Comparison of the thermodynamic properties of the complexes formed by structurally similar aminoglycosides indicated that the 2'- and the 6'-amino groups of the substrates are involved in binding to the enzyme. Thermodynamic properties of the complexes formed by aminoglycosides differing only at the 3'-hydroxyl group suggested that the absence of this group does not alter the thermodynamic parameters of the ternary APH(3')-IIIa-metal-ATP-aminoglycoside complex. Our results also indicate that protonation of ligand and protein ionizable groups is coupled to the complex formation between aminoglycosides and APH(3')-IIIa. Comparison of DeltaH values for different aminoglycoside-enzyme complexes indicates that enzyme and substrates undergo significant conformational changes in complex formation.


Backbone resonance assignments of a promiscuous aminoglycoside antibiotic resistance enzyme; the aminoglycoside phosphotransferase(3')-IIIa
Serpersu, Engin H.; Özen, Can; Norris, Adrianne L.; Steren, Carlos; Whittemore, Neil (2010-04-01)
The aminoglycoside phosphotransferase(3')-IIIa (APH) is a promiscuous enzyme and renders a large number of structurally diverse aminoglycoside antibiotics useless against infectious bacteria. A remarkable property of this similar to 31 kDa enzyme is in its unusual dynamic behavior in solution; the apo-form of the enzyme exchanges all of its backbone amide protons within 15 h of exposure to D (2) O while aminoglycoside-bound forms retain similar to 40% of the amide protons even after > 90 h of exposure. More...
Studies of enzymes that cause resistance to aminoglycosides antibiotics.
Serpersu, Engin H; Özen, Can; Wright, Edward (2008-01-01)
Aminoglycoside antibiotics are highly potent, wide-spectrum bactericidals (1, 2). Bacterial resistance to aminoglycosides, however, is a major problem in the clinical use of aminoglycosides. Enzymatic modification of aminoglycosides is the most frequent resistance mode among several resistance mechanisms employed by resistant pathogens (1,3). Three families of aminoglycoside modifying enzymes, O-phosphotransferases, N-acetyltransferases, and N-nucleotidyltransferases, are known to have more than 50 enzymes ...
Impact of untreated hospital effluents on dissemination of antibiotic resistance genes
Kayalı, Osman; İçgen, Bülent; Department of Biotechnology (2019)
Hospital wastewaters (HWWs) were reported to be hotspots for antibiotics and antibiotic-resistant bacteria. However, little is known about the impact of these effluents on the dissemination of antibiotic-resistance genes (ARGs). In this study, therefore, HWWs were monitored for 16S rRNA gene for overall bacterial genes and seven ARGs of aadA, tetA, cmlA, sul1, qnrS, ermB and blaCTX-M corresponding to commonly used antibiotics aminoglycosides, tetracyclines, amphenicols, sulfonamides, quinolones, macrolide-l...
Thermodynamics and Kinetics of Association of Antibiotics with the Aminoglycoside Acetyltransferase (3)-IIIb, a Resistance-Causing Enzyme
Norris, Adrianne L.; Özen, Can; Serpersu, Engin H. (2010-05-18)
The thermodynamic and kinetic properties of interactions of antibiotics with the aminoglycoside acetyltransferase (3)-IIIb (AAC) are determined with several experimental methods. These data represent the first such characterization of an enzyme that modifies the 2-deoxystreptamine ring common to all aminoglycoside antibiotics. Antibiotic substrates For AAC include kanamycin A, kanamycin B, tobramycin, sisomicin, neomycin B, paromomycin, lividomycin A, and ribostamycin. Kinetic studies show that kanamycin gr...
Thermostabilization of panomycocin, a novel exo-beta-1,3-glucanase isolated from pichia anomala NCYC 434, by using excipients and computational methods
Muhammed, Muhammed Tilahun; İzgü, Kadri Fatih; Son, Çağdaş Devrim; Department of Biomedical Engineering (2015)
As the risks for fungal infections increased, the prevalence of invasive fungal infections increased. Therefore, the demand for antifungal agents has risen. Moreover, the currently used antifungal agents have serious side effects and resistance development resulting from their mechanisms of action. Thus, novel antifungal agents with mechanisms that will not affect the host mammalian cells are in need. Yeast killer proteins which are naturally occuring toxins are good candidates for such types of agents. Pan...
Citation Formats
C. Özen, “Thermodynamics of aminoglycoside binding to aminoglycoside-3 ’-phosphotransferase IIIa studied by isothermal titration calorimetry,” BIOCHEMISTRY, pp. 14667–14675, 2004, Accessed: 00, 2020. [Online]. Available: