Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

• AadA6 from P. aeruginosa confers antibiotic resistance to sensitive E. coli strain.
• AadA6 at concentrations >100 μM equilibrates between monomeric and dimeric forms.
• Deletion of 17 residues from the carboxy-terminus decreases enzymatic activity.
• At 1 mM MgCl2 there is a strong substrate inhibition effect by streptomycin.
• A model of the central ternary complex suggests a mode of action of AadA6.

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl2 for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl2. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.

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