Aminoglycoside Resistance Resulting from Tight Drug Binding to an Altered Aminoglycoside Acetyltransferase

Open Access
Publisher Website
Google Scholar
Abstract
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 μM) and a weaker affinity for amikacin ( K d , ≈60 μM). 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.

This publication has 32 references indexed in Scilit: