title: Novel aminoglycoside 2''-phosphotransferase identified in a gram-negative pathogen.
abstract: Aminoglycoside 2″-phosphotransferases are the major aminoglycoside-modifying enzymes in clinical isolates of enterococci and staphylococci. We describe a novel aminoglycoside 2″-phosphotransferase from the Gram-negative pathogen Campylobacter jejuni, which shares 78% amino acid sequence identity with the APH(2″)-Ia domain of the bifunctional aminoglycoside-modifying enzyme aminoglycoside (6') acetyltransferase-Ie/aminoglycoside 2″-phosphotransferase-Ia or AAC(6')-Ie/APH(2″)-Ia from Gram-positive cocci, which we called APH(2″)-If. This enzyme confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested. Steady-state kinetic studies demonstrated that GTP, and not ATP, is the preferred cosubstrate for APH(2″)-If. The enzyme phosphorylates the majority of 4,6-disubstituted aminoglycosides with high catalytic efficiencies (k(cat)/K(m) = 10(5) to 10(7) M(-1) s(-1)), while the catalytic efficiencies against the 4,6-disubstituted antibiotics amikacin and isepamicin are 1 to 2 orders of magnitude lower, due mainly to the low apparent affinities of these substrates for the enzyme. Both 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates of APH(2″)-If, but are inhibitors. The antibiotic susceptibility and substrate profiles of APH(2″)-If are very similar to those of the APH(2″)-Ia phosphotransferase domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
authors.forename: Marta
authors.initials: M
authors.lastname: Toth
authors.forename: Hilary
authors.initials: H
authors.lastname: Frase
authors.forename: Nuno T
authors.initials: NT
authors.lastname: Antunes
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
mesh_terms: D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C048564:aminoglycoside 2''-phosphotransferase