Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

AbstractThe 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 5mM MgCl2 for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1mM 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 (>100μM). 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

Linear Extrapolation of Missing Radiographic Progression Scores Does Not Spuriously overestimate overall Radiographic Progression in Rheumatoid Arthritis

Pathophysiology and treatment of rheumatic disease