Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase.
The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(6')-Iy, from Salmonella enterica confers resistance toward a number of aminoglycoside antibiotics. The structural gene was cloned and expressed and the purified enzyme existed in solution as a dimer of ca. 17 000 Da monomers. Acetyl-CoA was the preferred acyl donor, and most therapeutically important aminoglycosides were substrates for acetylation. Exceptions are those aminoglycosides that possess a 6'-hydroxyl substituent (e.g., lividomycin). Thus, the enzyme exhibited regioselective and exclusive acetyltransferase activity to 6'-amine-containing aminoglycosides. The enzyme exhibited Michaelis-Menten kinetics for some aminoglycoside substrates but "substrate activation" with others. Kinetic studies supported a random kinetic mechanism for the enzyme. The enzyme was inactivated by iodoacetamide in a biphasic manner, with half of the activity being lost rapidly and the other half more slowly. Tobramycin, but not acetyl-CoA, protected against inactivation. Each of the three cysteine residues (C70, C109, C145) in the wild-type enzyme were carboxamidomethylated by iodoacetamide. Cysteine 109 in AAC(6')-Iy is conserved in 12 AAC(6') enzyme sequences of the major class I subfamily. Surprisingly, mutation of this residue to alanine neither abolished activity nor altered the biphasic inactivation by iodoacetamide. The maximum velocity and V/K values for a number of aminoglycosides were elevated in this single mutant, and the kinetic behavior of substrates exhibiting linear vs nonlinear kinetics was reversed. Cysteine 70 in AAC(6')-Iy is either a cysteine or a threonine residue in all 12 AAC(6') enzymes of the major class I subfamily. The double mutant, C109A/C70A, was not inactivated by iodoacetamide. The double mutant exhibited large increases in the K(m) values for both acetyl-CoA and aminoglycoside substrates, and all aminoglycoside substrates exhibited Michaelis-Menten kinetics. Solvent kinetic isotope effects on V/K were normal for the WT enzyme and inverse for the double mutant. We discuss a chemical mechanism and the likely rate-limiting steps for both the wild-type and mutant forms of the enzyme.
Magnet
S
S
Unité des Agents Antibactériens, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France.
Lambert
T
T
Courvalin
P
P
Blanchard
J S
JS
D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002876:Chromosomes, Bacterial / Q000201:enzymology* / Q000235:genetics*; D017666:Deuterium Oxide / Q000737:chemistry; D004352:Drug Resistance, Microbial / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D019779:Salmonella enterica / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D012997:Solvents; D013379:Substrate Specificity / Q000235:genetics
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D012997:Solvents; D000123:Acetyltransferases; C056532:aminoglycoside N(6')-acetyltransferase; D017666:Deuterium Oxide; D007460:Iodoacetamide