title: Large-scale preparation, purification, and crystallization of UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase from Escherichia coli.
abstract: The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli, an enzyme involved in the biosynthesis of the bacterial peptidoglycan monomer unit, was overproduced and purified to homogeneity on a large scale, yielding 4 mg of protein per liter of bacterial culture. Crystals of the complex with the substrate UDP-MurNAc-L-Ala were grown by the hanging drop method using ammonium sulfate as the precipitant. They are tetragonal with cell dimensions a = b = 65.5 A and c = 134.59 A, space group P4(1) or P4(3), and contain one monomer of 46,842 Da in the asymmetric unit. In order to use the multiple-wavelength anomalous diffraction method for phasing, a selenomethionine derivative of the protein has also been overproduced, purified, and crystallized.
authors.affiliation: Unité de Recherche Associée 1131, Centre National de la Recherche Scientifique, Biochimie Moléculaire et Cellulaire, Université de Paris-Sud, Orsay, France.
authors.forename: G
authors.initials: G
authors.lastname: Auger
authors.forename: L
authors.initials: L
authors.lastname: Martin
authors.forename: J
authors.initials: J
authors.lastname: Bertrand
authors.forename: P
authors.initials: P
authors.lastname: Ferrari
authors.forename: E
authors.initials: E
authors.lastname: Fanchon
authors.forename: S
authors.initials: S
authors.lastname: Vaganay
authors.forename: Y
authors.initials: Y
authors.lastname: Pétillot
authors.forename: J
authors.initials: J
authors.lastname: van Heijenoort
authors.forename: D
authors.initials: D
authors.lastname: Blanot
authors.forename: O
authors.initials: O
authors.lastname: Dideberg
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012645:Selenomethionine / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D012645:Selenomethionine; D010453:Peptide Synthases; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase