Pyridine nucleotide cycle of Salmonella typhimurium: regulation of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase.

Nicotinic acid phosphoribosyl transferase (NAPRTase) and nicotinamide deamidase activities from Salmonella typhimurium were examined regarding their regulation by either feedback inhibition or repression mechanisms. The results indicate that neither enzyme is subject to feedback inhbition. Nicotinamide deamidase does not appear to be under repression control. NAPRTase, however, is repressed when cells are grown in minimal medium supplemented with various intermediates of the pyridine nucleotide cycle. The concentration of exogenously supplied pyridine nucleotide necessary to effect repression of NAPRTas was found to be that concentration which will result in a nadA mutant generation time of less than 60 min. Furthermore, the results presented indicate that nicotinamide adenine dinucleotide is the actual corepressor molecule. The analogs 6-aminonicotinic acid and 6-aminonicotinamide were also capable of repressing NAPRTase, but only when an intact pyridine nucleotide cycl permitted conversion to 6-aminonicotinamide adenine dinucleotide. The role of a repressible NAPRTase is discussed in relation to the overall functioning of the pyridine nucleotide cycle.     

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 analog interference with metabolic processes in mitogen-stimulated human T lymphocytes

The differential metabolic effects of three nicotinamide analogs, 6-aminonicotinamide, 3-aminobenzamide, and 5-methylnicotinamide, were analyzed in mitogen-stimulated preparations of human T lymphocytes. Mitogen stimulation with the phorbol ester TPA and a monoclonal antibody to the T3 cell surface antigen caused an increase in cellular NAD and ATP levels and a marked increase in glucose metabolism as demonstrated by an increase in cellular levels of glucose 6-phosphate and a sevenfold increase in radioactive CO2 formation from [l-14C]glucose. 6-Aminonicotinamide had drastic inhibitory effects on the mitogen-stimulated increases in NAD and ATP levels as well as on the metabolism of glucose. Treatment of the mitogen-stimulated cells with 6-aminonicotinamide also caused a marked increase in cellular levels of 6-phosphogluconate, suggesting inhibition of the hexose monophosphate shunt at 6-phosphogluconate dehydrogenase. Radioactive CO2 formation from [6-14C]glucose showed that metabolism through the tricarboxylic acid cycle was not used to compensate for the inhibition of the hexose monophosphate shunt pathway. Treatment of cells with 3-aminobenzamide had the opposite effect of 6-aminonicotinamide in that cellular NAD levels increased, presumable due to inhibition of poly(ADP-ribose) polymerase. 3-Aminobenzamide did not interfere with ATP or glucose 6-phosphate levels and did not cause significant elevations of 6-phosphogluconate. Thus, 6-aminonicotinamide appears to have direct inhibitory effects on the synthesis of both pyridine nucleotides and poly(ADP-ribose), whereas 3-aminobenzamide has its major inhibitory effect on poly(ADP-ribose) synthesis. 5-Methylnicotinamide also interferes with the mitogen-stimulated increase in NAD levels but not as effectively as 6-aminonicotinamide. The alterations in pyridine nucleotide metabolism resulting from treatment with these nicotinamide analogs can produce drastic and diverse alterations in pathways of glucose utilization and energy generation.     

6-Aminonicotinamide-resistant mutants of Salmonella typhimurium

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.     

Pyridine nucleotide cycle of Salmonella typhimurium: in vivo recycling of nicotinamide adenine dinucleotide

The relative contribution of the two known pyridine nucleotide cycles of Salmonella typhimurium towards the intracellular recycling of nicotinamide adenine dinucleotide was determined. The results indicate that intracellular nicotinamide adenine dinucleotide is recycled by both the four-membered pyridine nucleotide cycle (PNC IV) and the six-membered pyridine nucleotide cycle (PNC VI) with a relative contribution of 60 to 69% and 31 to 40%, respectively. These studies also revealed a nicotinic acid mononucleotide-degradative activity which converts nicotinic acid mononucleotide to nicotinic acid. This represents the first demonstration of a functional PNC IV pathway in S. typhimurium.     

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.     

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.     

Thin-layer chromatographic separation of intermediates of the pyridine nucleotide cycle

A rapid thin-layer chromatographic procedure for separation of the compounds comprising the intermediates in the salvage pathway known as the pyridine nucleotide cycle plus quinolinic acid and the reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate is described. The method utilizes silica gel high-performance thin-layer plates and a mobile phase of methanol, tetrabutylammonium hydroxide, and acetonitrile. The time required for analysis is greatly reduced and results in greater than 96% purity of each migrating compound.     

Pentose cycle flux and fatty acid synthesis in bovine adipose tissue slices incubated with 6-aminonicotinamide

The effects of the purported inhibitor of 6-phosphogluconate dehydrogenase, 6-aminonicotinamide, on lipogenesis from acetate and the metabolism of glucose were investigated in bovine adipose tissue. The incorporation of [U-14C]acetate and tritium from [3-3H]glucose into fatty acids was stimulated by 6-aminonicotinamide proportionately, indicating that the pentose cycle provided the same percentage of NADPH required for fat synthesis in the absence and presence of 6-aminonicotinamide. Tissue samples incubated with 6-aminonicotinamide displayed higher maximal activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase than control samples. The cellular content of 6-phosphogluconate was increased by 6-aminonicotinamide by 40% in samples incubated with 2 mM glucose (plus 33 mU/ml insulin) and 10 mM acetate; 6-aminonicotinamide stimulated the production of L-lactate in either the absence or presence of acetate. Studies with 1-, 6-, and U-14C-labeled glucose indicated that 6-aminonicotinamide increased the proportion of utilized glucose metabolized by the pentose cycle in the absence, but not in the presence of acetate. Unlike results observed in rat adipose tissue, the primary effect of 6-aminonicotinamide was to increase the proportion of NADPH produced by the pentose cycle that was utilized for fat synthesis secondarily to the stimulation of lipogenesis by an unknown mechanism.     

Biosynthesis of pyridine alkaloids from Tripterygium wilfordii

Nicotinic acid-6-¹⁴C and nicotinamide adenine dinucleotide-carbonyl-¹⁴C were rapidly metabolized in T. wilfordii Hook. with formation of all compounds in the pyridine nucleotide cycle. Nicotinic acid-6-¹⁴C and the nicotinamide moiety of NAD were efficiently incorporated into wilfordic acid and hydroxywilfordic acid, the pyridinium moieties of the ester alkaloids. The structures of wilfordic acid and hydroxywilfordic acid were confirmed using GLC-MS. The molecular formulae of the four isolated alkaloids were determined by high resolution MS and agreed with earlier results based on elemental analysis.     

Modification of aspartate before its condensation with dihydroxyacetone phosphate during quinolinic acid formation in Escherichia coli.

A crude enzyme preparation from a nadA mutant of Escherichia coli was used to catalyze the conversion of [14C]aspartic acid into a precursor of quinolinic acid, a key intermediate in the biosynthesis of nicotinamide adenine dinucleotide.     

Thiosulfate- and Sulfide-Dependent Pyridine Nucleotide Reduction and Gluconeogenesis in Intact Thiobacillus neapolitanus

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

Metabolism of nicotinic acid in plant cell suspension cultures, III: Formation and metabolism of trigonelline (author's transl)]

Cell suspension cultures of Phaseolus aureus, Glycinemax., Cicer arietinum and Chenopodium rubrum convert nicotinic acid and nicotinamide into N-methyl nicotinic acid (trigonelline). Application of [carboxyl-14C]- and [N-methyl-14C]nicotinic acid to cell cultures demonstrated that 1) the nicotinic acid moiety of trigonelline is funnelled into the pyridine nucleotide cycle, 2) trigonelline is demethylated partly oxidatively, but predominantly non-oxidatively, transferring the methyl carbon atom to still unknown acceptors, and 3) uptake of trigonelline by mung bean cell cultures is accompanied by demethylation and instantaneous remethylation reactions. Cell suspension cultures of parsley (Petroselinum hortense Hoffm.) show uptake but no metabolism of trigonelline. The data are compared with trigonelline metabolism in intact plants.     

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.     

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.     

Adenine cycle in hepatopancreocytes of Helix pomatia (Gastropoda)

Intact hepatopancreocytes were obtained from hibernating or active purinotelic snails, H. pomatia (Gastropoda). When incubated with [14C]glycine or [14C]formate, they synthesized de novo purine compounds, including also adenylates, adenosine and adenine. Hepatopancreocytes resynthesized also adenylates and other purine compounds from [3H]adenine or from [3H]adenosine split by the H. pomatia cell enzyme to adenine; the resynthesis of ADP+ATP was proportional to adenine concentration. Thus all reactions of the postulated adenine cycle: AMP leads to adenosine leads to adenine leads to AMP occur in the intact hepatopancreocytes; this cycle could probably be responsible for maintenance of the high level of adenylates during winter sleep.     

Utilization and metabolism of NAD by Haemophilus parainfluenzae

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the beta-configuration of the glycopyridine linkage. (4) An intact carboxamide groups is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.     

Biosynthetic preparation of [riboflavin-2-14C]flavin adenine dinucleotide using Clostridium kluyveri

The biosynthetic preparation of [riboflavin-2-(14)C]flavin adenine dinucleotide from extracellular [2-(14)C]riboflavin by a growing culture of Clostridium kluyveri, first reported by Decker and coworkers, has been implemented using new media and more convenient isolation procedures.