Open AccessN ‐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
Author(s) -
Gil Fernando,
RamónMaiques Santiago,
Marina Alberto,
Fita Ignacio,
Rubio Vicente
Publication year - 1999
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444999005351
Subject(s) - escherichia coli , recombinant dna , monomer , cloning (programming) , enzyme , biochemistry , plasmid , gene , crystallization , microbiology and biotechnology , biology , kinase , chemistry , crystallography , stereochemistry , organic chemistry , computer science , programming language , polymer
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 µmol s −1 mg −1 ), lacks Met1 and forms dimers (shown by cross‐linking). Crystals of unliganded NAGK diffract to 2 Å and belong to space group P 6 1 22 or its enantiomorph P 6 5 22 (unit‐cell parameters a = b = 78.6, c = 278.0 Å) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 Å and belong to space group C 222 1 (unit‐cell parameters a = 60.0, b = 71.9, c = 107.4 Å), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.