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

Escherichia coil NAD synthetase was overexpressed and purified to homogenei ty. The recombinant protein was active in an in vitro enzyme assay. The enz yme required approximately 1.5 mM magnesium for optimal activity. The pH op timum 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 ad enine dinucleotide and adenosine triphosphate under similar conditions. The se crystals diffract to 2.0-Angstrom resolution and belong to trigonal spac e group P3(1)21 with unit cell dimensions of a = b = 91.766, c = 74.17 Angs trom and alpha = beta 90 degrees, gamma = 120 degrees. The structure of the complex has been determined using the molecular replacement method, (C) 19 99 Academic Press.