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")'s 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"'-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.
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")'s 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"'-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.
Authors: Mateusz P Czub; Brian Zhang; M Paul Chiarelli; Karolina A Majorek; Layton Joe; Przemyslaw J Porebski; Alina Revilla; Weiming Wu; Daniel P Becker; Wladek Minor; Misty L Kuhn Journal: Biochemistry Date: 2018-12-12 Impact factor: 3.162
Authors: Nishad Thamban Chandrika; Keith D Green; Jacob L Houghton; Sylvie Garneau-Tsodikova Journal: ACS Med Chem Lett Date: 2015-09-30 Impact factor: 4.345