Prodigious substrate specificity of AAC(6′)-APH(2′ʹ), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci Original Research Article

Background High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6′)-APH(2″), an enzyme with 6′-N-acetyltransferase and 2″-O-phosphotransferase activities. The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents. Results In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6′)-APH(2″) in Bacillus subtilis. The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities. Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6′)-APH(2″)ʹs dramatic negative impact on clinical aminoglycoside therapy. Both lividomycin A and paromomycin, aminoglycosides lacking a 6′-amino group, were acetylated by AAC(6′)-APH(2″). The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation. Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3′-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3′-OH and the 3″ʹ-OH of ring D, both unprecedented observations for this enzyme. Furthermore, the phosphorylation site of lividomycin A was determined to be the 5″-OH of the pentose ring C. Conclusions The bifunctional AAC(6′)-APH(2″) has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups. The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design.