Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan

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Abstract
The relationship between the acetylation of peptidoglycan and that of aminoglycosides in Providencia stuartii has been investigated both in vivo and in vitro. Adaptation of the assay for peptidoglycan N-->O-acetyltransferase permitted an investigation of the use of peptidoglycan as a source of acetate for the N acetylation of aminoglycosides by gentamicin N-acetyltransferase [EC 2.3.1.59; AAC(2')]. The peptidoglycan from cells of P. stuartii PR50 was prelabelled with 3H by growth in the presence of N-[acetyl-3H]glucosamine. Under these conditions, [3H]acetate was confirmed to be transferred to the C-6 position of peptidoglycan-bound N-acetylmuramyl residues. Isolated cells were subsequently incubated in the presence of various concentrations of gentamicin and tobramycin (0 to 5x MIC). Analysis of various cellular fractions from isolated cells and spent culture medium by the aminoglycoside-binding phosphocellulose paper assay revealed increasing levels of radioactivity associated with the filters used for whole-cell sonicates of cells treated with gentamicin up to 2 x MIC. Beyond this concentration, a decrease in radioactivity was observed, consistent with the onset of cell lysis. Similar results were obtained with tobramycin, but the increasing trend was less obvious. The transfer of radiolabel to either aminoglycoside was not observed with P. stuartii PR100, a strain that is devoid of AAC(2')-Ia. A high-performance anion-exchange chromatography-based method was established to further characterize the AAC(2')-Ia-catalyzed acetylation of aminoglycosides. The high-performance liquid chromatography (HPLC)-based method resolved a tobramycin preparation into two peaks, both of which were collected and confirmed by 1H nuclear magnetic resonance to be the antibiotic. Authentic standards of 2'-N-acetyltobramycin were prepared and were well separated from the parent antibiotic when subjected to the HPLC analysis. By applying this technique, the transfer of radiolabelled acetate from the cell wall polymer peptidoglycan to tobramycin was confirmed. In addition, isolated and purified AAC(2')-Ia was shown to catalyze in vitro the transfer of acetate from acetyl-coenzyme A, soluble fragments of peptidoglycan, and N-acetylglucosamine to tobramycin. These data further support the proposal that AAC(2')-Ia from P. stuartii may have a physiological role in its secondary metabolism and that its activity on aminoglycosides is simply fortuitous.