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 amino-glycoside 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 Km 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.
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