The chromosomal gene aac(6')-Ii, encoding an aminoglycoside 6'-N-acetyltransferase in Enterococcus faecium, renders this organism resistant to moderate levels of many aminoglycoside antibiotics. The ubiquitous presence of aac(6')-Ii in E. faecium complicates the selection of antibiotics for treatment of infections caused by this organism. In view of the importance of this enzyme, we have initiated studies to gain an understanding of its molecular mechanism of acetyl transfer. The AAC(6')-Ii enzyme was overexpressed in Escherichia coli and purified in a simple three-step procedure which yields 55 mg of pure dimeric protein per liter of cell culture. Steady-state kinetic analyses revealed a broad substrate specificity and demonstrated that acetylation occurs exclusively at position N-6'. k(cat)/Km values were on the order of 10(4) M(-1) s(-1), which is relatively low compared to other aminoglycoside-modifying enzymes. In addition, MIC values were positively correlated with k(cat), the rate when the enzyme is saturated with the aminoglycoside substrate, and not with k(cat)/Km, the rate at low aminoglycoside (sub-Km) concentrations. These results describe an enzyme which is not optimally evolved for aminoglycoside inactivation and suggest that this chromosomally encoded enzyme may have an alternate physiological function.