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

J Biol Chem. 2003 Aug 29;278(35):33049-55. doi: 10.1074/jbc.M302257200. Epub 2003 May 27.


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.

MeSH terms

  • Amide Synthases / chemistry*
  • Amide Synthases / metabolism*
  • Aminohydrolases / chemistry*
  • Ammonia / chemistry
  • Anthranilate Synthase*
  • Binding Sites
  • Catalysis
  • Escherichia coli / metabolism
  • Genetic Complementation Test
  • Glutamine / metabolism
  • Models, Chemical
  • Models, Molecular
  • Nitrogenous Group Transferases / chemistry*
  • Oligonucleotides / chemistry
  • Oxidation-Reduction
  • Phenotype
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Structure, Tertiary
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Sulfhydryl Compounds / chemistry
  • Thermotoga maritima


  • Oligonucleotides
  • Recombinant Proteins
  • Sulfhydryl Compounds
  • Glutamine
  • Ammonia
  • Nitrogenous Group Transferases
  • Aminohydrolases
  • nitrilase
  • Anthranilate Synthase
  • anthranilate synthase, glutamine amidotransferase subunit
  • Amide Synthases
  • NAD+ synthase
  • Qns1 protein, S cerevisiae