The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad

Nicotinamide-adenine dinucleotide (NAD(+)) synthetases catalyze the last step in NAD(+) metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD(+) synthetase converts nicotinic acid adenine dinucleotide to NAD(+) via an adenylylated intermediate. All of the known eukaryotic NAD(+) synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD(+) synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD(+) synthetase, Qns1. Independently, human NAD(+) synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD(+) synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD(+) synthetase and this was speculated largely to mediate NAD(+) synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD(+) synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.