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The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo-protein, followed by addition of an aqueous solution of CoCl(2). Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6(5), with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X-ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co-substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86-Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40-Nδ(1), Cys78-Sγ, His81-Nδ(1), and 86Met-Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)-substituted pseudoazurin.

The copper(II) centre of the blue copper protein pseudoazurin fromAlcaligenes faecalishas been substituted by zinc(II)viadenaturing the protein, chelation and removal of copper and refolding the apoprotein, followed by the addition of an aqueous solution of ZnCl2. Vapour-diffusion experiments produced colourless hexagonal crystals (space groupP65), which when cryocooled had unit-cell parametersa=b= 49.01,c= 98.08 Å. Diffraction data collected at 100 K using a copper sealed tube were phased by the weak anomalous signal of five S atoms and one Zn atom. The structure was fitted manually and refined to 1.6 Å resolution. The zinc-substituted protein exhibits similar overall geometry to the native structure with copper. Zn2+binds more strongly to its four ligand atoms (His40 Nδ1, Cys78 Sγ, His81 Nδ1and Met86 Sδ) and retains the tetrahedral arrangement, although the structure is less distorted than the native copper protein.

An NAD(+)-dependent psychrophilic alcohol dehydrogenase (ADH) from the Antarctic psychrophile Moraxella sp. TAE123 has been purified to homogeneity. The enzyme consists of four identical subunits, each containing two Zn ions. Protein crystals suitable for X-ray diffraction were obtained under optimized salting-out crystallization conditions using ammonium sulfate as a precipitating agent. The crystals are hexagonal bipyramids and belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = 136.4, c = 210.7 A. They contain one protein homotetramer in the asymmetric unit. Diffraction data were collected to 2.2 A under cryogenic conditions using synchrotron radiation.

The 3D structure of the apo-pseudoazurin (copper free pseudoazurin) from Alcaligenes faecalis strain S-6 is determined and refined at pH 6.7 using X-ray diffraction data to 1.85 A resolution. The final crystallographic R-factor is 0.164. Comparing the structures of apo-pseudoazurin and the native (Cu2+) protein, we observed limited differences ranging between 0.1-0.4 A at the vicinity of the copper site, at the loops connecting the secondary structural elements, at certain beta-strands and at the amino and carboxy termini of the protein.

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.