The molecular basis of the expansive substrate specificity of the antibiotic resistance enzyme aminoglycoside acetyltransferase-6'-aminoglycoside phosphotransferase-2". The role of ASP-99 as an active site base important for acetyl transfer.
The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6'-aminoglycoside phosphotransferase-2" (AAC(6')- aminoglycoside phosphotransferase-2", capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6')-Ie, is the only member of the large AAC(6') subclass known to modify fortimicin A and catalyze O-acetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6')-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6')-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.
Boehr
David D
DD
Antimicrobial Research Centre, Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
Jenkins
Stephen I
SI
Wright
Gerard D
GD
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D001665:Binding Sites; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; C108453:6'-aminoglycoside acetyltransferase-2''-aminoglycoside phosphotransferase; D000123:Acetyltransferases; D017853:Phosphotransferases (Alcohol Group Acceptor)