title: Eukaryotic NAD+ synthetase Qns1 contains an essential, obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase.
abstract: 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 A from one active site to another within Qns1 monomers.
authors.affiliation: Structural Biology and Bioinformatics Program, Kimmel Cancer Center, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Pawel
authors.initials: P
authors.lastname: Bieganowski
authors.forename: Helen C
authors.initials: HC
authors.lastname: Pace
authors.forename: Charles
authors.initials: C
authors.lastname: Brenner
mesh_terms: D019732:Amide Synthases / Q000737:chemistry* / Q000378:metabolism*; D000619:Aminohydrolases / Q000737:chemistry*; D000641:Ammonia / Q000737:chemistry; D000878:Anthranilate Synthase; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D005973:Glutamine / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D019884:Nitrogenous Group Transferases / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D020124:Thermotoga maritima
chemical_list: D009841:Oligonucleotides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D005973:Glutamine; D000641:Ammonia; D019884:Nitrogenous Group Transferases; D000619:Aminohydrolases; C021867:nitrilase; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit; D019732:Amide Synthases; C038479:NAD+ synthase; C477525:Qns1 protein, S cerevisiae