Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

Abstract

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.

Keywords: AMPCPP, α,β-methyleneadenosine 5′-triphosphate; Aminoglycoside adenylyltransferase; Antibiotic modification; Circular dichroism; Enzyme kinetics; Homology modelling; MALS, Multi-angle light scattering; MIC, Minimum Inhibitory Concentration; Multi-angle light scattering; [1-264]AadA6, genetically truncated AadA6 at the carboxy-terminus; [1-281]AadA6 or AadA6, full length aminoglycoside adenylyltransferase.

Graphical abstract

Fig. 1

Circular Dichroism spectropolarimetry data and thermal denaturation curves. (A) Far-UV CD spectra of AadA6 (red) and [1-264]AadA6 (blue) were recorded at 10 °C. (B) Superimposed thermal melting CD curves for both proteins are labelled as in (A). The ordinates of both plots present values of mean residue ellipticity [θ].

Fig. 2

Multi-angle light scattering (MALS) curves. Relative quasi-elastic light scattering intensities (right axis) of AadA6 (red) and [1-264]AadA6 (blue) in 50 mM Tris–HCl pH 7.5 buffer at 1 mM MgCl₂ as function of their elution volumes from an analytical gel-filtration column. The two peaks correspond to the dimer and monomer of each protein. The estimates of the respective molecular hydrodynamic radii (R_H) as function of volume slice are also shown. The plots were produced by the data analysis software provided by the instrument manufacturer.

Fig. 3

Theoretical modelling of AadA6 structure. Active site view with both ATP and streptomycin. The crystal structures used as templates were, AadA from S. enterica (PDB: 4CS6) and kanamycin nucleotidyltransferase from S. aureus (PDB: 1KNY). The labelled protein residues that interact with the substrates are shown in stick representation (atom colours, carbon: yellow for streptomycin and light yellow for ATP, nitrogen: blue, oxygen: red, phosphorus: orange). The sphere in magenta denotes the magnesium ion. The dashes indicate H-bonding or the co-ordination of the magnesium ion. The figure was prepared with PyMOL (http://www.pymol.org).