Eukaryotic NAD+ synthetase Qns1 contains an essential, obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase

NAD(+) is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl) ation. NAD(+) synthetase catalyzes the final step in NAD(+) synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD(+) synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD(+) synthetases are ammonia-dependent. Earlier, we discovered that glutamine-dependent NAD(+) synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD(+) synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD(+) synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD(+) synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 Angstrom from one active site to another within Qns1 monomers.