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...
TUL MUNNA, SADIA; Akdağ, Akın; Çulfaz Emecen, Pınar Zeynep; Department of Polymer Science and Technology (2021-9-10)
The polymeric membranes for antimicrobial applications are in high demand nowadays, since the bacteria had developed the resistance against the antibiotics. The membrane technology could become an efficient tool to cope with the antibiotic resistant bacteria which are becoming an unevitable problem on the health and pocket of a common man. To address this issue, the current research is carried out to synthesize polymer with antibacterial properties. Norbornene and oxonorborne monomers with special modificat...
Thermostability and regulation of Clostridium thermocellum L-lactate dehydrogenase expressed in Escherichia coli
Assa, P; Ozkan, M; Özcengiz, Gülay (2005-01-01)
In this study, L-lactate dehydrogenase (L-LDH) of Clostridium thermocellum previously cloned aid expressed in Escherichia coli FMJ39 was partially purified and characterised. Optimum temperature and pH of the enzyme were found as 50 degrees C and 7.5, respectively. Different concentrations of Mn2+ did not affect the enzyme activity. Addition of 20-30 mM Mg2+, or the other hand, increased the LDH activity by about 10%. Relatively high concentrations of NaCl (2 M), fructose-1,6-diphosphate (FDP, 5 mM), ATP (1...
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: