Structural study of Escherichia coli NAD synthetase: Overexpression, purification, crystallization, and preliminary crystallographic analysis

Journal Article

Escherichia coli NAD synthetase was over-expressed 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-Å resolution and belong to trigonal space group P3121 with unit cell dimensions of a = b = 91.766, c = 74.17 Å and α = β = 90°, γ = 120°. The structure of the complex has been determined using the molecular replacement method.

Full Text

Duke Authors

Cited Authors

  • Ozment, C; Barchue, J; Delucas, LJ; Chattopadhyay, D

Published Date

  • 1999

Published In

Volume / Issue

  • 127 / 3

Start / End Page

  • 279 - 282

International Standard Serial Number (ISSN)

  • 1047-8477

Digital Object Identifier (DOI)

  • 10.1006/jsbi.1999.4152