Novel Aminoglycoside 2 ''-Phosphotransferase Identified in a Gram-Negative Pathogen

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.