Quantitation of NAD⁺ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

Abstract

Nicotinamide adenine dinucleotide (NAD+) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD+ decomposition products. NAD+ biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD+ biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD+ and NADH (the reduced form of NAD+) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1Δ), and NAD+ de novo pathway knockout (qpt1Δ) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5 % glucose (CR). We have utilized 14C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and 14C detection to quantitate the total amounts of NAD+ and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1Δ yeast exclusively utilize extracellular nicotinic acid for NAD+ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD+ concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD+:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1Δ strains, while no increase in lifespan is observed for the npt1Δ strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD+ or NADH for lifespan extension under CR.

Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae

“…Yeast cells appear to produce NAD + predominantly via the NA/Nam salvage pathway during exponential growth (27) because standard growth media contain high concentrations (400 μg/l or ~ 3 μM) of niacin (NA and Nam) and budding yeast has a very efficient NA transporter Tna1 (Km for NA = ~ 1.7 μM) (17). Interestingly, although NR is an endogenous metabolite (28), standard yeast growth media do not contain NR because they do not support the growth of NAD + synthesis mutants defective in both de novo and NA/Nam salvaging (for example, the qns1 Δ mutant).…”

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

“…NMN is generated by phosphorylating nicotinamide riboside (NR), which is catalyzed by the kinase Nrk1 at the expense of ATP (9,10). It has been shown that yeast cells produce NAD ϩ predominantly via the NA/Nam salvage pathway during exponential growth (11). More recently, NR has been shown to be an efficient NAD ϩ precursor that contributes to the NAD ϩ pool and supports NAD ϩ -dependent reactions (9,10).…”

Secretion of Quinolinic Acid, an Intermediate in the Kynurenine Pathway, for Utilization in NAD + Biosynthesis in the Yeast Saccharomyces cerevisiae

“…Electrospray ionization was performed in positive-ion mode. Intracellular concentration of NAD ϩ was calculated by taking the volume of a haploid BY4742 cell to be 70 m 3 , the volume of a kynurenine pathway-or salvage pathway-knockout cell to be 40 m 3 (23), and the A 600 of 0.1 to correspond to 0.7 ϫ 10 6 cells/ml. LC-MS analysis for QA.…”