REGIOSPECIFICITY OF AMINOGLYCOSIDE PHOSPHOTRANSFERASE FROM ENTEROCOCCI AND STAPHYLOCOCCI (APH(3')-IIIA)

The broad-spectrum aminoglycoside phosphotransferase, APH(3')-IIIa, co nfers resistance to several aminoglycoside antibiotics in opportunisti c pathogens of the genera Staphylococcus and Enterococcus. The profile of the drug resistance phenotype suggested that the enzyme would tran sfer a phosphate group from ATP to the 3'-hydroxyl of aminoglycosides. In addition, resistance to the 3'-deoxyaminoglycoside antibiotic, liv idomycin A, suggested possible transfer to the 5 ''-hydroxyl of the ri bose [Trieu-Cuot, P., & Courvalin, P. (1983) Gene 23, 331-341]. Using purified overexpressed enzyme, we have prepared and purified the produ cts of APH(3')-IIIa-dependent phosphorylation of several of aminoglyco side antibiotics. Mass spectral analysis revealed that 4,6-disubstitut ed aminocyclitol antibiotics such as amikacin and kanamycin are monoph osphorylated, while 4,5-disubstituted aminoglycosides such as butirosi n A, ribostamycin, and neomycin B are both mono- and diphosphorylated by APH(3')-IIIa. Using a series of one- and two-dimensional H-1, C-13, and P-31 NMR experiments, we have unambiguously assigned the regiospe cificity of phosphoryl transfer to several antibiotics. The 4,6-disubs tituted aminocyclitol antibiotics are exclusively phosphorylated at th e 3'-OH hydroxyl, and the 4,5-disubstituted aminocyclitol antibiotics can be phosphorylated at both the 3'- and 5 ''-hydroxyls. The first ph osphorylation can occur on either the 3'- or 5 ''-hydroxyl group of ne omycin B or butirosin A. Initial phosphotransfer to the 3'-position pr edominates for butirosin while the 5 ''-OH is favored for neomycin. Th ese results open the potential for the rational design of aminoglycosi de kinase inhibitors based on functionalization of either the 6-aminoh exose or the pentose rings of aminoglycoside antibiotics.