PMID:10544053

Citation	Ozment, C, Barchue, J, DeLucas, LJ and Chattopadhyay, D (1999) Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis. J. Struct. Biol. 127:279-82
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.
Links	PubMed Online version:10.1006/jsbi.1999.4152
Keywords	Amide Synthases/chemistry; Amide Synthases/genetics; Amide Synthases/isolation & purification; Amino Acid Sequence; Cloning, Molecular; Computer Graphics; Crystallization; Crystallography, X-Ray; Escherichia coli/enzymology; Models, Molecular; Molecular Sequence Data; Peptide Fragments/chemistry; Protein Conformation; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification
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