Towards structural understanding of feedback control of arginine biosynthesis: cloning and expression of the gene for the arginine-inhibited N-­acetyl-L-glutamate kinase from Pseudomonas aeruginosa, purification and crystallization of the recombinant enzyme and preliminary X-ray studies

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the second step in the pathway of arginine biosynthesis in microorganisms and plants. In many species, it is the pathway-controlling enzyme and is subject to feedback inhibition by arginine. The gene for the best characterized arginine-inhibitable NAGK, that from Pseudomonas aeruginosa, has been cloned in a pET22 plasmid and overexpressed in Escherichia coli. The enzyme was purified in three steps to 95% purity and was shown by cross-linking to form dimers. It was crystallized by the hanging-drop vapour-diffusion method at 277 K in the presence of ADP, Mg and N-­acetyl-L-glutamate. The crystallization solution contained 0.1 M sodium cacodylate pH 6.5, 150–170 mM magnesium acetate and 13% polyethylene glycol 8000. Prismatic crystals of maximum dimension approximately 0.5 mm diffract to 2.75 Å resolution and belong to space group P1 (unit-cell parameters a = 71.86, b = 98.78, c = 162.9 Å, α = 91.49, β = 92.03, γ = 107.56°). Packing density considerations agree with 6–18 NAGK monomers in the asymmetric unit, with a corresponding solvent content of 79–36%. Self-rotation function calculations confirm the space group and suggest the presence of 3–7 dimers in the unit cell.