Glutamine-dependent NAD+ Synthetase

Abstract

Glutamine-dependent NAD؉ synthetase, Qns1, utilizes a glutamine aminotransferase domain to supply ammonia for amidation of nicotinic acid adenine dinucleotide (NaAD ؉ ) to NAD ؉ . Earlier characterization of Qns1 suggested that glutamine consumption exceeds NAD ؉ production by 40%. To explore whether Qns1 is systematically wasteful or whether additional features account for this behavior, we performed a careful kinetic and molecular genetic analysis. In fact, Qns1 possesses remarkable properties to reduce waste. The glutaminase active site is stimulated by NaAD ؉ more than 50-fold such that glutamine is not appreciably consumed in the absence of NaAD ؉ . Glutamine consumption exceeds NAD؉ production over the whole range of glutamine and NaAD ؉ substrate concentrations with greatest efficiency occurring at saturation of both substrates. Kinetic data coupled with site-directed mutagenesis of amino acids in the predicted ammonia channel indicate that NaAD ؉ stimulates the glutaminase active site in the k cat term by a synergistic mechanism that does not require ammonia utilization by the NaAD ؉ substrate. Six distinct classes of Qns1 mutants that fall within the glutaminase domain and the synthetase domain selectively inhibit components of the coordinated reaction.Glutamine-dependent NAD ϩ synthetase Qns1 (1) is one of many enzymes that couples a glutamine amidotransferase (GAT) 2 domain to a second active site that requires ammonia gas as a reactant (2-4). The second active site of Qns1 is an NAD ϩ synthetase domain that reacts nicotinic acid adenine dinucleotide (NaAD ϩ ) with ATP to form an activated, adenylylated intermediate (NaAD-AMP), releasing pyrophosphate. In turn, NaAD-AMP is attacked by ammonia to produce NAD ϩ plus AMP. Ideally, the enzyme would produce one equivalent of NAD ϩ and AMP for every glutamine hydrolyzed to glutamate. This ideal stoichiometry, depicted in Fig. 1, has been attributed to glutamine-dependent NAD ϩ synthetase since the first description of the enzyme by Preiss and Handler (5). The simplicity of Fig. 1 notwithstanding, characterization of the native (6) and recombinant (1) purified enzyme has never been entirely consistent with ideality. Whether the enzyme is, in fact, capable of perfect stoichiometry, potentially at a "sweet spot" of substrate concentrations, has not been investigated.Though eukaryotic and some prokaryotic NAD ϩ synthetases have long been understood to be glutamine-dependent enzymes (5, 7), the GAT domain of these enzymes initially escaped recognition as a glutaminase because it is neither a triad-type GAT, an N-terminal nucleophile-type GAT (2), nor one related to aminoacyl-tRNA transamidases (8). In the course of characterizing the domain structure of members of the nitrilase superfamily, we observed that the glutamine dependence of all eukaryotic and particular prokaryotic NAD ϩ synthetases could be accounted for by the presence of a putative GAT domain related to nitrilase (9, 10). Site-directed mutagenesis of yeast Qns1 coupled to biochemical and physiologi...

NAD+ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity

“…Thus, steady state levels of the substrates of NaMN/NMN adenylyltransferases were detected under all conditions measured. In contrast, NaAD, the product of NaMN adenylylation and the substrate of glutamine-dependent NAD + synthetase Qns1 [35], was below 1% of the level of NAD + under all conditions examined, suggesting that NaAD produced from the de novo or Preiss-Handler pathways is driven forward under k cat / K m conditions for Qns1 [36]. …”

The Vitamin Nicotinamide: Translating Nutrition into Clinical Care

“…Initially, nicotinamide is changed to its mononucleotide form (NMN) with the enzyme nicotinic acid/nicotinamide adenylyltransferase yielding the dinucleotides NAAD + and NAD + . NAAD + also yields NAD + through NAD + synthase [6] or NAD + can be synthesized through nicotinamide riboside kinase that phosphorylates nicotinamide riboside to NMN [7,8]. These cellular pathways are essential for energy metabolism and may directly impact normal physiology, as well as disease progression [9,10,11,12].…”

NAD(P) Biosynthesis Enzymes as Potential Targets for Selective Drug Design

“…Both classes display a highly conserved C-terminal domain, responsible for the NADS activity, including a P-loop motif characteristic of the N-type family of ATP pyrophosphatases [150]. The glutaminedependent enzymes possess an additional N-terminal nitrilase-like domain that hydrolyzes glutamine and channels the released ammonia to the NADS domain [151][152][153]. The nitrilase domain contains a conserved catalytic triad formed by a Cys nucleophile and two ion-paired Glu and Lys residues [151].…”

“…The nitrilase domain contains a conserved catalytic triad formed by a Cys nucleophile and two ion-paired Glu and Lys residues [151]. Interestingly, the glutaminase activity of the nitrilase domain is highly dependent on the binding of NaAD on NADS domain, preventing wasteful substrate consumption [153]. The glutamine-dependent enzymes can also use ammonia as nitrogen donor to NADS domain, albeit generally with much lower affinity [68].…”