title: Glutamate-5-kinase from Escherichia coli: gene cloning, overexpression, purification and crystallization of the recombinant enzyme and preliminary X-ray studies.
abstract: Glutamate-5-kinase (G5K) catalyzes the first step of proline (and, in mammals, ornithine) biosynthesis. It is a key regulatory point of these routes, since it is the subject of feedback allosteric inhibition by proline or ornithine. The Escherichia coli gene (proB) for G5K was cloned in pET22, overexpressed in E. coli, purified in a few steps in high yield to 95% homogeneity in the highly active proline-inhibitable form and was shown by cross-linking to be a tetramer. It was crystallized by the hanging-drop vapour-diffusion method at 294 K in the presence of ADP, MgCl(2) and L-glutamate using 1.6 M MgSO(4), 0.1 M KCl in 0.1 M MES pH 6.5 as the crystallization solution. The tetragonal bipyramid-shaped crystals diffracted to 2.5 A resolution using synchrotron radiation. The crystals belong to space group P4(1(3))2(1)2, with unit-cell parameters a = b = 101.1, c = 178.6 A, and contain two monomers in the asymmetric unit, with 58% solvent content.
authors.affiliation: Fundación Valenciana de Investigaciones Biomédicas (FVIB), Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Pérez-Arellano
authors.forename: Fernando
authors.initials: F
authors.lastname: Gil-Ortiz
authors.forename: Javier
authors.initials: J
authors.lastname: Cervera
authors.forename: Vicente
authors.initials: V
authors.lastname: Rubio
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase