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 A resolution and belong to space group P1 (unit-cell parameters a = 71.86, b = 98.78, c = 162.9 A, alpha = 91.49, beta = 92.03, gamma = 107.56 degrees ). 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.
Fernández-Murga
M Leonor
ML
Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Científicas (IBV-CSIC), Jaime Roig 11, Valencia 46010, Spain.
Ramón-Maiques
Santiago
S
Gil-Ortiz
Fernando
F
Fita
Ignacio
I
Rubio
Vicente
V
D001120:Arginine / Q000096:biosynthesis*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D025461:Feedback, Physiological; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D001120:Arginine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase