title: Revisiting the nucleotide and aminoglycoside substrate specificity of the bifunctional aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2'')-Ia enzyme.
abstract: The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Hilary
authors.initials: H
authors.lastname: Frase
authors.forename: Marta
authors.initials: M
authors.lastname: Toth
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000617:Aminoglycosides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000502:physiology; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000123:Acetyltransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C048564:aminoglycoside 2''-phosphotransferase