Aminoglycoside resistance resulting from tight drug binding to an altered aminoglycoside acetyltransferase.
The aacA29b gene, which confers an atypical aminoglycoside resistance pattern to Escherichia coli, was identified on a class 1 integron from a multidrug-resistant isolate of Pseudomonas aeruginosa. On the basis of amino acid sequence homology, it was proposed that the gene encoded a 6'-N-acetyltransferase. The resistance gene was cloned into the pET23a(+) vector, and overexpression conferred high-level resistance to the usual substrates of the aminoglycoside N-acetyltransferase AAC(6')-I, except netilmicin. The level of resistance conferred by aacA29b correlated perfectly with the level of expression of the gene. The corresponding C-terminal six-His-tagged AAC(6')-29b protein was purified and found to exist as a dimer in solution. With a spectrophotometric assay, an extremely feeble AAC activity was detected with acetyl coenzyme A (acetyl-CoA) as an acetyl donor. Fluorescence titrations of the protein with aminoglycosides demonstrated the very tight binding of tobramycin, dibekacin, kanamycin A, sisomicin (K(d), </=1 micro M) and a weaker affinity for amikacin (K(d), approximately 60 micro M). The binding of netilmicin and acetyl-CoA could not be detected by either fluorescence spectroscopy or isothermal titration calorimetry. The inability of AAC(6')-29b to efficiently bind acetyl-CoA is supported by an alignment analysis of its amino acid sequence compared with those of other AAC(6')-I family members. AAC(6')-29b lacks a number of residues involved in acetyl-CoA binding. These results lead to the conclusion that AAC(6')-29b is able to confer aminoglycoside resistance by sequestering the drug as a result of tight binding.
Magnet
Sophie
S
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Smith
Terry-Ann
TA
Zheng
Renjian
R
Nordmann
Patrice
P
Blanchard
John S
JS
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects; D008969:Molecular Sequence Data
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000123:Acetyltransferases; C019681:aminoglycoside acetyltransferase