title: Mechanistic characterization of the bifunctional aminoglycoside-modifying enzyme AAC(3)-Ib/AAC(6')-Ib' from Pseudomonas aeruginosa.
abstract: A recently discovered bifunctional antibiotic-resistance enzyme named AAC(3)-Ib/AAC(6')-Ib', from Pseudomonas aeruginosa, catalyzes acetylation of aminoglycoside antibiotics. Since both domains are acetyltransferases, each was cloned and purified for mechanistic studies. The AAC(3)-Ib domain appears to be highly specific to fortimicin A and gentamicin as substrates, while the AAC(6')-Ib' domain exhibits a broad substrate spectrum. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism. The use of dead-end and product inhibition and solvent-isotope effect reveals that both domains catalyze their reactions by a steady-state ordered Bi-Bi kinetic mechanism, in which acetyl-CoA is the first substrate that binds to the active site, followed by binding of the aminoglycoside antibiotic. Subsequent to the transfer of the acetyl group, acetylated aminoglycoside is released prior to coenzyme A. The merger of two genes to create a bifunctional enzyme with expanded substrate profile would appear to be a recent trend in evolution of resistance to aminoglycoside antibiotics, of which four examples have been documented in the past few years.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Choonkeun
authors.initials: C
authors.lastname: Kim
authors.forename: Adriel
authors.initials: A
authors.lastname: Villegas-Estrada
authors.forename: Dusan
authors.initials: D
authors.lastname: Hesek
authors.forename: Shahriar
authors.initials: S
authors.lastname: Mobashery
mesh_terms: D000617:Aminoglycosides / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000617:Aminoglycosides; D017931:DNA Primers; D004798:Enzymes