Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl-¹⁴C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [¹⁴C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-¹⁴C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.     

Trigonelline biosynthesis and the pyridine nucleotide cycle in Coffea arabica fruits: Metabolic fate of [carboxyl-C]nicotinic acid riboside

Trigonelline is a major component in coffee seeds and may contribute to the bitter taste of the resultant beverage. To determine the trigonelline biosynthetic pathway in coffee fruits, we investigated the metabolic fate of [carboxyl-¹⁴C]nicotinic acid riboside and in situ activity of related enzymes. Exogenously supplied [carboxyl-¹⁴C]nicotinic acid riboside was rapidly converted to nicotinic acid mononucleotide and was utilized for NAD synthesis. Nicotinic acid riboside was also used for trigonelline synthesis, but this process took longer than NAD synthesis. These results indicate that an efficient nicotinic acid riboside salvage system functions in coffee fruits, and that trigonelline is synthesized mainly from nicotinic acid produced by the degradation of NAD.     

Pyridine nucleotide biosynthesis in Claviceps

Claviceps purpurea grown on synthetic medium incorporated labeled [7-¹⁴]nicotinic acid and [7-¹⁴C]nicotinamide into NaMN, des-NAD, NAD, and NADP. Label also appeared in NMN and N-methyl nicotinamide. The specific activities of NAD, NADP, and NMN are compatible with the operation of the Preiss-Handler pathway of NAD biosynthesis (nicotinic acid → NaMN → des-NAD → NAD → NADP). The relative amounts of NaMN:des-NAD:NAD and NADP were about 8:1:36:10 on incubation of Claviceps with nicotinic acid for 6 hr. The incorporation of nicotinamide into NAD proceeds mainly by conversion to nicotinic acid catalyzed by nicotinamide deamidase.Tryptophan ([U-¹⁴C]benzene ring) was incorporated into NAD demonstrating the presence of the tryptophan-nicotinic acid pathway. No qualitative difference in pyridine nucleotide intermediates was noted in C. purpurea CPM, which does not produce clavine alkaloids, and Claviceps 47A which does produce clavine alkaloids.     

Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [³H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD → nicotinamide mononucleotide → nicotinamide riboside → nicotinic acid riboside → nicotinic acid mononucleotide → nicotinic acid adenine dinucleotide → NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-¹⁴C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-¹⁴C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.     

Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza

We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots.     

Metabolism of nicotinamide, adenine and inosine in developing microspore-derived canola (Brassica napus) embryos

The metabolic fate of [carbonyl-(14)C]nicotinamide, [8-(14)C]adenine and [8-(14)C]inosine was examined in microspore-derived canola (Brassica napus) embryos at different developmental stages: globular stage (day 10, stage 1), early cotyledonary stage (day 20, stage 2), late cotyledonary stage (day 25, stage 3), and fully developed stage (day 35, stage 4). Uptake of [8-(14)C]nicotinamide by the embryos was always rapid. A lower uptake rate was found for [8-(14)C]adenine and [8-(14)C]inosine, especially at stages 1 and 2. [Carbonyl-(14)C]nicotinamide was converted to nicotinic acid and further metabolized to pyridine nucleotides (NAD/NADP). Some radioactivity was also associated to nicotinic acid glucoside. [8-(14)C]adenine was efficiently utilized for the synthesis of adenine nucleotides and RNA. A small fraction of adenine was degraded to CO(2) via ureides. Up to 40% of [8-(14)C]inosine was salvaged to nucleotides and RNA, although degradation of [8-(14)C]inosine to CO(2) was pronounced. At stage 1, highest salvage activities of nicotinamide, adenine and inosine were observed. In contrast, the lowest purine salvage and highest purine catabolism were found in stage 3 embryos. These results suggest that both nicotinamide and purine salvage for NAD/NADP and purine nucleotides synthesis are extremely high in the globular stage (stage 1). These activities decrease gradually until the late cotyledonary stage (stage 3), before increasing again in the fully developed embryos (stage 4). Overall it appears that nicotinamide and purine salvage are required in support of active growth during the initial phases of embryogenesis and at the end of the maturation period, in preparation for post-embryonic growth.     

Degradation of nicotinamide adenine dinucleotide in cell suspension cultures

Metabolites of [carbonyl-¹⁴C]-NAD in cell suspension cultures of mung bean, soybean and garbanzo bean are trigonelline and compounds of the pyridine nucleotide cycle. Degradation of nicotinate does not occur. In parsley cell cultures nicotinate degradation and formation of nicotinic acid N-α-l-arabinoside were observed. These conjugates are alternative reservoir forms of nicotinic acid. The adenine moiety of NAD is degraded in cell cultures via hypoxanthine-xanthine-allantoin-allantoic acid, with accumulation of the latter two compounds.     

Pyridine nucleotide cycle and trigonelline (N-methylnicotinic acid) synthesis in developing leaves and fruits of Coffea arabica

We examined the biosynthesis of trigonelline in leaves and fruits of Arabica coffee ( Coffea arabica ) plants. [³H]Quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide and [¹⁴C]nicotinic acid, which are degradation products of NAD, were converted to trigonelline and pyridine nucleotides. These tracer experiments suggest that the pyridine nucleotide cycle, nicotinamide → nicotinic acid → nicotinic acid mononucleotide (NaMN) → nicotinic acid adenine dinucleotide (NaAD) → NAD → nicotinamide mononucleotide (NMN) → nicotinamide, operates in coffee plants, and trigonelline is synthesized from nicotinic acid formed in the cycle. Trigonelline accumulated up to 18 µmol per leaf in developed young leaves, and then decreased with age. Although the biosynthetic activity of trigonelline from exogenously supplied [¹⁴C]nicotinamide was observed in aged leaves, the endogenous supply of nicotinamide may be limited, reducing the contents in these leaves. Trigonelline is synthesized and accumulated in fruits during development. The trigonelline synthesis in pericarps is much higher than that in seeds, but its content in seeds is higher than pericaps, so that some of the trigonelline synthesized in the pericarps may be transported to seeds. Trigonelline in seeds may be utilized during germination, as its content decreases. Trigonelline synthesis from [¹⁴C]nicotinamide was also found in Theobroma cacao plants, but instead of trigonelline, nicotinic acid-glucoside was synthesized from [¹⁴C]nicotinamide in Camellia sinensis plants.     

Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium

A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.     

Microbial NAD Metabolism: Lessons from Comparative Genomics

Summary: NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways.     

Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1

NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.     

Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5��-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD⁺, which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD⁺ metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5′-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5′-nucleotidase, and Sdt1, initially classified as a pyrimidine 5′-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD⁺. Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD⁺ metabolism.     

Effect of short-term salt stress on the metabolic profiles of pyrimidine, purine and pyridine nucleotides in cultured cells of the mangrove tree, Bruguiera sexangula

To investigate the short‐term (3 h) effect of salt on the metabolism of purine, pyrimidine and pyridine nucleotides in mangrove (Bruguiera sexangula) cells, we examined the uptake and overall metabolism of radiolabelled intermediates involved in the de novo pathways and substrates of salvage pathways for nucleotide biosynthesis in the presence and absence of 100 mM NaCl. Uptake by the cells of substrates for the salvage pathways was much faster than uptake of intermediates of the de novo pathways. The activity of the de novo pyrimidine biosynthesis estimated by [2‐¹⁴C]orotate metabolism was not significantly affected by the salt. About 20–30% of [2‐¹⁴C]uridine, [2‐¹⁴C]uracil and more than 50% of [2‐¹⁴C]cytidine were salvaged for pyrimidine nucleotide biosynthesis. However, substantial quantities of these compounds were degraded to ¹⁴CO₂ via β‐ureidopropionate (β‐UP), and degradation of β‐UP was increased by the salt. The activities of the de novo pathway, estimated by [2‐¹⁴C] 5‐aminoimidazole‐4‐carboxamide ribonucleoside, and the salvage pathways from [8‐¹⁴C]adenosine and [8‐¹⁴C]guanosine for the purine nucleotide biosynthesis were not influenced by the salt. Most [8‐¹⁴C]hypoxanthine was catabolised to ¹⁴CO₂, and other purine compounds are also catabolised via xanthine. Purine catabolism was stimulated by the salt. [³H]Quinolinate, [carbonyl‐¹⁴C]nicotinamide and [carboxyl‐¹⁴C]nicotinic acid were utilised for the biosynthesis of pyridine nucleotides. The salvage pathways for pyridine nucleotides were significantly stimulated by the salt. Trigonelline was synthesised from all pyridine precursors that were examined; its synthesis was also stimulated by the salt. We discuss the physiological role of the salt‐stimulated reactions of nucleotide metabolism.     

Nicotinamide riboside kinase structures reveal new pathways to NAD+

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD⁺) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD⁺ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD⁺.     

Nicotinamide Riboside Kinase Structures Reveal New Pathways to NAD+

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD⁺) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD⁺ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD⁺.     

Pyridine nucleotide cycle of Salmonella typhimurium: isolation and characterization of pncA, pncB, and pncC mutants and utilization of exogenous nicotinamide adenine dinucleotide.

Mutants of Salmonella typhimurium LT-2 deficient in nicotinamidase activity (pncA) or nicotinic acid phosphoribosyltransferase activity (pncB) were isolated as resistant to analogs of nicotinic acid and nicotinamide. Information obtained from interrupted mating experiments placed the pncA gene at 27 units and the pncB gene at 25 units on the S. typhimurium LT-2 linkage map. A major difference in the location of the pncA gene was found between the S. typhimurium and Escherichia coli linkage maps. The pncA gene is located in a region in which there is a major inversion of the gene order in S. typhimurium as compared to that in E. coli. Growth experiments using double mutants blocked in the de novo pathway to nicotinamide adenine dinucleotide (NAD) (nad) and in the pyridine nucleotide cycle (pnc) at either the pncA or pncB locus, or both, have provided evidence for the existence of an alternate recycling pathway in this organism. Mutants lacking this alternate cycle, pncC, have been isolated and mapped via cotransduction at 0 units. Utilization of exogenous NAD was examined through the use of [14C]carbonyl-labeled NAD and [14C]adenine-labeled NAD. The results of these experiments suggest that NAD is degraded to nicotinamide mononucleotide at the cell surface. A portion of this extracellular nicotinamide mononucleotide is then transported across the cell membrane by nicotinamide mononucleotide glycohydrolase and degraded to nicotinamide in the process. The remaining nicotinamide mononucleotide accumulates extracellularly and will support the growth of nadA pncB mutants which cannot utilize the nicotinamide resulting from the major pathway of NAD degradation. A model is presented for the utilization of exogenous NAD by S. typhimurium LT-2.     

Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

NAD⁺ levels in resting human lymphocytes obtained from 20 donors were found to be 69.9 ± 21.7 pmols/10⁶ cells. After 3 days of phytohemagglutinin (PHA) stimulation the NAD⁺ levels rose to 452 ± 198 pmols/10⁶ cells. NADH, NADP⁺ and NADPH also increased in mitogen-stimulated lymphocytes, but the major portion of the increase in total pyridine nucleotide pools was accounted for by the increase in NAD⁺. When PHA-stimulated lymphocytes were incubated in nicotinamide-deficient growth medium, there was no significant increase in their total pyridine nucleotide pools; however, the ratios of oxidized to reduced pyridine nucleotides changed in a similar fashion to cells grown in medium containing nicotinamide. When lymphocytes in nicotinamide-deficient medium were stimulated with PHA they increased their levels of DNA synthesis and cell replication in a similar fashion to cells growing in nicotinamide-supplemented media. Human lymphocytes were able to synthesize pyridine nucleotides from nicotinamide or nicotinic acid; however, in the absence of a preformed pyridine ring they did not efficiently use tryptophan for the synthesis of NAD. Uptake of [carbonyl-¹⁴C]nicotinamide and conversion to NAD was markedly increased in PHA-stimulated lymphocytes; these cells also showed a marked increase in activity of the enzyme adenosine-triphosphate-nicotinamide mononucleotide (ATP-NMN) adenylyl transferase.     

Preliminary evidence for a pyridine nucleotide cycle in Bordetella pertussis

Preliminary evidence that Bordetella pertussis has a functional pyridine nucleotide cycle was the observation that [¹⁴C]-nicotinic acid was rapidly metabolized during its uptake by the bacteria to pyridine nucleotides and nicotinamide. Nicotinamide deamidase activity, necessary for the completion of the cycle by conversion of nicotinamide to nicotinic acid, was found in a soluble extract (20000 x g supernatant) of B. pertussis cell lysates.This work was supported by the Science Research Council and Wellcome Research Laboratories.     

Assimilation of NAD(+) precursors in Candida glabrata

The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.