N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms.
The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.1 micromol s-1 mg-1), lacks Met1 and forms dimers (shown by cross-linking). Crystals of unliganded NAGK diffract to 2 A and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 A) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 A and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 A), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.
Instituto de Biomedicina de Valencia (CSIC), C/Jaime Roig 11, 46010-Valencia, Spain.
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F
Gil
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Ramón-Maiques
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A
Marina
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I
Fita
V
V
Rubio
D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase