title: Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis.
abstract: Escherichia coli NAD synthetase was overexpressed and purified to homogeneity. The recombinant protein was active in an in vitro enzyme assay. The enzyme required approximately 1.5 mM magnesium for optimal activity. The pH optimum was found to be 8.0-8.5. The recombinant protein was crystallized at room temperature using the hanging-drop vapor diffusion technique with 1.5 M lithium sulfate, 0. 1 M Hepes buffer at pH 7.5 as precipitant. The protein was also crystallized in the presence of its substrates, nicotinic acid adenine dinucleotide and adenosine triphosphate under similar conditions. These crystals diffract to 2.0-A resolution and belong to trigonal space group P3(1)21 with unit cell dimensions of a = b = 91.766, c = 74.17 A and alpha = beta = 90 degrees, gamma = 120 degrees. The structure of the complex has been determined using the molecular replacement method.
authors.affiliation: Division of Geographic Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
authors.forename: C
authors.initials: C
authors.lastname: Ozment
authors.forename: J
authors.initials: J
authors.lastname: Barchue
authors.forename: L J
authors.initials: LJ
authors.lastname: DeLucas
authors.forename: D
authors.initials: D
authors.lastname: Chattopadhyay
mesh_terms: D019732:Amide Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003196:Computer Graphics; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D019732:Amide Synthases; C038479:NAD+ synthase