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.