Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide adenine dinucleotide glycohydrolase, nicotinamide mononucleotide glycohydrolase, and nicotinamide adenine dinucleotide pyrophosphatase activities.

Extracts of Salmonella typhimurium were chromatographed by using Sephadex G-150 to separate the various enzymes involved with pyridine nucleotide cycle metabolism. This procedure revealed a previously unsuspected nicotinamide adenine dinucleotide (NAD) glycohydrolase (EC 3.2.2.5) activity, which was not observed in crude extracts. In contrast to NAd glycohydrolase, NAD pyrophosphatase (EC 3.6.1.22) was readily measured in crude extracts. This enzyme possessed a native molecular weight of 120,000. Other enzymes examined included nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.00), molecular weight of 43,000; NMN glycohydrolase (EC 3.2.2.14), molecular weight of 67,000; nicotinic acid phosphoribosyl transferase (EC 2.4.2.11), molecular weight of 47,000; and nicotinamide deamidase (EC 3.5.1.19), molecular weight of 35,000. NMN deamidase and NMN glycohydrolase activities were both examined for end product repression by measuring their activities in crude extracts prepared from cells grown with and without 10(-5) M nicotinic acid. No repression was observed with either activity. Both activities were also examined for feedback inhibition by NAD, reduced NAD, and NADP. NMN deamidase was unaffected by any of the compounds tested. NMN glycohydrolase was greatly inhibited by NAD and reduced NAD, whereas NADP was much less effective. Inhibition of NMN glycohydrolase was found to level off at an NAD concentration of ca. 1 mN, the approximate intracellular concentration of NAD.     

Metabolism of nicotinamide mononucleotide in beef liver

Nicotinamide mononucleotide (NMN) is not only an intermediate for the biosynthesis but also a degradation product of pyridine cofactors in animal tissues. Among the animal tissues tested, the highest NMN catabolizing activity was detected in beef liver (5.6 mumol/min/g tissue). This activity was 16 times higher than the NAD hydrolysis catalyzed by the liver NAD glycohydrolase. As a result of enzymatic analysis of the NMN splitting process, two types of enzyme responsible for this catabolism were partially purified and identified as a membrane-bound 5'-nucleotidase and a cytoplasmic nicotinamide riboside (NR) phosphorylase. No specific NMN glycohydrolase could be found in contrast to results observed in bacterial systems. The 5'-nucleotidase and NR phosphorylase constitute an obligatory process of the pyridine nucleotide cycle. The dephosphorylation and phosphorolysis catalyzed suggest that these enzymes could serve as an important mechanism for salvaging the ribose and nicotinamide moieties of NMN and pyridine nucleotides in the cell and a process that could be regulated at the mononucleotide level by this "NMN cycle" rather than by a NAD glycohydrolase cycle. In addition to the enzymatic properties of these enzymes, a regulatory mechanism by nucleotides such as ATP was also demonstrated.     

NAD metabolism in Vibrio cholerae.

Extracts of Vibrio cholerae were assayed for various enzymatic activities associated with pyridine nucleotide cycle metabolism. The activities measured include NAD glycohydrolase, nicotinamide deamidase, nicotinamide mononucleotide deamidase, and nicotinic acid phosphoribosyltransferase. The results obtained demonstrate the existence in V. cholerae of the five-membered pyridine nucleotide cycle and the potential for a four-membered pyridine nucleotide cycle. The data presented also suggest that most of the NAD glycohydrolase in V. cholerae extracts is not directly related to cholera toxin.     

Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family

The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.     

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.     

Genetic characterization of the pnuC gene, which encodes a component of the nicotinamide mononucleotide transport system in Salmonella typhimurium.

The pnuC gene, which encodes a component of the nicotinamide mononucleotide transport system, has been mapped and oriented. The gene order of the pnuC region, which is at min 17 of the Salmonella chromosome, is nadA-pnuC-aroG-gal. Polarity tests, with pnuC::Mu d-lac operon fusions, reveal that the pnuC gene is the promoter distal gene in an operon with the nadA gene, which encodes the second enzyme of the pyridine biosynthetic pathway. The nadA pnuC operon is regulated by the NadI repressor. The pnuC gene also has its own promoter, since strains with a nadA::Tn10d(Tc) insertion still express the pnuC gene at a low, unregulated level.     

The pyridine nucleotide cycle: presence of a nicotinamide mononucleotide-specific amidohydrolase in Propionibacterium shermanii

A specific nicotinamide mononucleotide amidohydrolase which catalyzes the stoichiometric conversion of NMN to nicotinate mononucleotide and ammonia has been partially purified from an extract of $\text{Propionibacterium}\text{shermanii}$. The reaction has optimum activity at pH 5.6, a K_m of 70 μM, and an experimental activation energy of 14.5 Kcal/mole. The enzyme appears to be highly specific for NMN. Neither free nicotinamide nor NAD, NADH, NADP, NADPH compete with NMN. Numerous substances such as isonicotinic acid hydrazide and quinolinic acid are also without effect. It can be stored at ?15° in 12% glycerol, but is somewhat unstable in the absence of this solvent. The enzyme is composed of a heatstable and a heat-sensitive subunit. This enzyme considerably simplifies the pyridine nucleotide cycle, and may, besides this salvage function for NAD, play a role in B₁₂ biosynthesis and in the bacterial DNA ligase reaction.     

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.     

Structure and Mechanism of the Bifunctional CinA Enzyme from Thermus thermophilus

CinA is a widely distributed protein in Gram-positive and Gram-negative bacteria. It is associated with natural competence and is proposed to have a function as an enzyme participating in the pyridine nucleotide cycle, which recycles products formed by non-redox uses of NAD. Here we report the determination of the crystal structure of CinA from Thermus thermophilus, in complex with several ligands. CinA was shown to have both nicotinamide mononucleotide deamidase and ADP-ribose pyrophosphatase activities. The crystal structure shows an unusual asymmetric dimer, with three domains for each chain; the C-terminal domain harbors the nicotinamide mononucleotide deamidase activity, and the structure of a complex with the product nicotinate mononucleotide suggests a mechanism for deamidation. The N-terminal domain belongs to the COG1058 family and is associated with the ADP-ribose pyrophosphatase activity. The asymmetry in the CinA dimer arises from two alternative orientations of the COG1058 domains, only one of which forms a contact with the KH-type domain from the other chain, effectively closing the active site into, we propose, a catalytically competent state. Structures of complexes with Mg²⁺/ADP-ribose, Mg²⁺/ATP, and Mg²⁺/AMP suggest a mechanism for the ADP-ribose pyrophosphatase reaction that involves a rotation of the COG1058 domain dimer as part of the reaction cycle, so that each active site oscillates between open and closed forms, thus promoting catalysis.     

Identification of a novel human nicotinamide mononucleotide adenylyltransferase

The enzyme nicotinamide mononucleotide adenylyltransferase is an ubiquitous enzyme catalyzing an essential step in NAD (NADP) biosynthetic pathway. In human cells, the nuclear enzyme, which we will now call NMNAT-1, has been the only known enzyme of this type for over 10 years. Here we describe the cloning and expression of a human cDNA encoding a novel 34.4kDa protein, that shares significant homology with the 31.9kDa NMNAT-1. We propose to call this enzyme NMNAT-2. Purified recombinant NMNAT-2 is endowed with NMN and nicotinic acid mononucleotide adenylyltransferase activities, but differs from NMNAT-1 with regard to chromosomal and cellular localization, tissue-specificity of expression, and molecular properties, supporting the idea that the two enzymes might play distinct physiological roles in NAD homeostasis.     

Metabolic design for selective production of nicotinamide mononucleotide from glucose and nicotinamide

β-Nicotinamide mononucleotide (NMN) is, one of the nucleotide compounds, a precursor of NAD⁺ and has recently attracted attention as a nutraceutical. Here, we develop a whole-cell biocatalyst using Escherichia coli, which enabled selective and effective high production of NMN from the inexpensive feedstock substrates glucose and nicotinamide (Nam). Notably, we identify two actively functional transporters (NiaP and PnuC) and a high-activity key enzyme (Nampt), permitting intracellular Nam uptake, efficient conversion of phosphoribosyl pyrophosphate (PRPP; supplied from glucose) and Nam to NMN, and NMN excretion extracellularly. Further, enhancement of the PRPP biosynthetic pathway and optimization of individual gene expression enable drastically higher NMN production than reported thus far. The strain extracellularly produces 6.79 g l⁻¹ of NMN from glucose and Nam, and the reaction selectivity from Nam to NMN is 86%. Our approach will be promising for low-cost, high-quality industrial production of NMN and other nucleotide compounds using microorganisms.     

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 nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica

Salmonella enterica can obtain pyridine from exogenous nicotinamide mononucleotide (NMN) by three routes. In route 1, nicotinamide is removed from NMN in the periplasm and enters the cell as the free base. In route 2, described here, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and the produced nicotinamide ribonucleoside (NmR) enters the cell via the PnuC transporter. Internal NmR is then converted back to NMN by the NmR kinase activity of NadR. Route 3 is seen only in pnuC* transporter mutants, which import NMN intact and can therefore grow on lower levels of NMN. Internal NMN produced by either route 2 or route 3 is deamidated to nicotinic acid mononucleotide and converted to NAD by the biosynthetic enzymes NadD and NadE.     

Importance of nicotinamide as an NAD precursor in the human erythrocyte

The effect of variation in the concentration of inorganic phosphate and of the pyridine precursors nicotinamide (NAm) and nicotinic acid (NA) on pyridine nucleotide synthesis was studied using intact human erythrocytes. A wide range of incubation times was employed. The results showed that under physiological conditions the rate of synthesis of NAD from NAm exceeded that from NA twofold, while the reverse situation pertained at higher and unphysiological substrate levels. The two pathways had different regulation points. For NAm the rate-limiting factor was the initial step, namely its conversion into the mononucleotide, while for NA it lay at the second step, conversion of NA mononucleotide (NAMN) to its adenine dinucleotide. At physiological substrate levels the uptake of NA and conversion to NAMN were rapid, while the uptake and conversion of NAm were time dependent. This process was stimulated significantly by inorganic phosphate only for NAm. These results indicate that while NA is the predominant precursor of human erythrocyte NAD at high (unphysiological) substrate and phosphate levels, NAm is more efficient as an NAD precursor under physiological conditions, suggesting an important and hitherto unrecognized role for nicotinamide in NAD synthesis in vivo.     

Assay of nicotinamide deamidase activity using high-performance liquid chromatography

A rapid, simple and reproducible method has been developed for the determination of nicotinamide deamidase activity using high-performance liquid chromatography (HPLC). Nicotinic acid (NA) liberated from nicotinamide (NAA) after a 15-min enzyme reaction was determined directly by HPLC without further separation steps. Both NA, the product, and NAA, the substrate were separated by reversed-phase ion-pair isocratic chromatography and detected at 261 nm. The present method could be applied to the measurement of deamidase activity in crude cell extracts prepared from several bacterial strains. The Michaelis constant of nicotinamide deamidase in Enterobacter agglomerans was 36 μM for NAA. This method is useful for the measurement of nicotinamide deamidase from various sources.     

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.     

Genetic characterization of pyridine nucleotide uptake mutants of Salmonella typhimurium

Two classes of pyridine nucleotide uptake mutants isolated previously in a strain of Salmonella typhimurium defective in both de novo NAD biosynthesis (nad) and pyridine nucleotide recycling (pncA) were analysed in terms of their genetic relationship to each other and their roles in the transport of nicotinamide mononucleotide as a precursor to NAD. The first class of uptake mutants, pnuA (99 units), failed to grow on nicotinamide mononucleotide (NMN) as a precursor for NAD. The second class, pnuB, grew on lower than normal levels of NMN and suppressed pnuA mutations. A third class of uptake mutant, pnuC, isolated in a nadB pncA pnuB background, also failed to grow on NMN. Transport studies and enzyme analyses confirmed these strains as defective in NMN uptake. A fourth locus, designated pnuD, was found to diminish NMN utilization in a nad pncA+ background. Tn10 insertions near pnuA, pnuC and pnuD were isolated and utilized in mapping studies. pnuA was found to map between thr and serB near trpR. The pnuC locus was cotransducible with nadA at 17 units while pnuD mapped at approximately 60 units. The biochemical and genetic data suggest that the pnuA and pnuC gene products cooperate in the utilization of NMN under normal conditions. A pnuB mutant, however, does not require the pnuA gene product for NMN uptake but does rely on the pnuC product. Fusion studies indicate that pnuC is regulated by internal NAD concentrations.     

NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5'-nucleotidase activity. The e (P4) protein is also shown to have NMN 5'-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.     

A novel preparation of nicotinamide mononucleotide

Nicotinamide mononucleotide is conveniently prepared from nicotinamide adenine dinucleotide by specific hydrolysis of the pyrophosphate bond using the Zr4+ ion as catalyst.