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

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.

Duke Scholars

Author Caroline Pinson Ozment Pediatrics, Critical Care Medicine