OVEREXPRESSION AND CHARACTERIZATION OF THE CHROMOSOMAL AMINOGLYCOSIDE6'-N-ACETYLTRANSFERASE FROM ENTEROCOCCUS-FAECIUM

The chromosomal gene aac(6')-Ii, encoding an aminoglycoside 6'-N-acety ltransferase in Enterococcus faecium, renders this organism resistant to moderate levels of many aminoglycoside antibiotics. The ubiquitous presence of aac(6')-li in E. faecium complicates the selection of anti biotics for treatment of infections caused by this organism, In view o f 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 overespressed in Escherichia coil and purified in a s imple three-step procedure which yields 55 mg of pure dimeric protein per liter of cell culture, Steady-state kinetic analyses revealed a br oad substrate specificity and demonstrated that acetylation occurs exc lusively at position N-6', k(cat)/K-m 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 ffere positively correlate d with k(cat), the rate when the enzyme is saturated with the aminogly coside substrate, and not with K-cat/l(km), the rate at low aminoglyco side (sub-K-m) concentrations. These results describe an enzyme which is not optimally evolved for aminoglycoside inactivation and suggest t hat this chromosomally encoded enzyme may have an alternate physiologi cal function.