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Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis.
J Struct Biol. 1999 Oct; 127(3):279-82.JS

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.

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Authors+Show Affiliations

Ozment C
Division of Geographic Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
Barchue J
No affiliation info available
DeLucas LJ
No affiliation info available
Chattopadhyay D
No affiliation info available

MeSH

Amide SynthasesAmino Acid SequenceCloning, MolecularComputer GraphicsCrystallizationCrystallography, X-RayEscherichia coliModels, MolecularMolecular Sequence DataPeptide FragmentsProtein ConformationRecombinant Proteins

Pub Type(s)

Journal Article

Language

eng

PubMed ID

10544053

Citation

Ozment, C, et al. "Structural Study of Escherichia Coli NAD Synthetase: Overexpression, Purification, Crystallization, and Preliminary Crystallographic Analysis." Journal of Structural Biology, vol. 127, no. 3, 1999, pp. 279-82.
Ozment C, Barchue J, DeLucas LJ, et al. Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis. J Struct Biol. 1999;127(3):279-82.
Ozment, C., Barchue, J., DeLucas, L. J., & Chattopadhyay, D. (1999). Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis. Journal of Structural Biology, 127(3), 279-82.
Ozment C, et al. Structural Study of Escherichia Coli NAD Synthetase: Overexpression, Purification, Crystallization, and Preliminary Crystallographic Analysis. J Struct Biol. 1999;127(3):279-82. PubMed PMID: 10544053.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis. AU - Ozment,C, AU - Barchue,J, AU - DeLucas,L J, AU - Chattopadhyay,D, PY - 1999/11/2/pubmed PY - 1999/11/2/medline PY - 1999/11/2/entrez SP - 279 EP - 82 JF - Journal of structural biology JO - J Struct Biol VL - 127 IS - 3 N2 - 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. SN - 1047-8477 UR - https://www.unboundmedicine.com/medline/citation/10544053/Structural_study_of_Escherichia_coli_NAD_synthetase:_overexpression_purification_crystallization_and_preliminary_crystallographic_analysis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1047-8477(99)94152-7 DB - PRIME DP - Unbound Medicine ER -