NADP and NAD utilization in Haemophilus influenzae

Exogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy-dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5'-nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD-nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5'-nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild-type H. influenzae strains, when NADP was used as the sole source of nicotinamide-ribosyl.     

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.     

Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.     

The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism

Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3?? resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships.     

Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae

Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.     

Lipoprotein e (P4) of Haemophilus influenzae: role in heme utilization and pathogenesis

Lipoprotein e (P4) of Haemophilus influenzae is a phosphomonoesterase, encoded by the hel gene, that has been implicated in the acquisition of heme by this fastidious organism. However, lipoprotein e (P4) is also involved in the utilization of NAD and NMN. Some reports have concluded that the reported heme-related growth defect actually reflects a growth defect for NAD. In the current study, hel insertion mutants were constructed and a role for e (P4) in heme acquisition was demonstrated independent of its role in NAD or NMN acquisition. In addition, a rat model of infection demonstrated a role for e (P4) in the pathogenesis of invasive disease.     

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.     

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.     

Weak coupling of ATP hydrolysis to the chemical equilibrium of human nicotinamide phosphoribosyltransferase

Human nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PP i) from nicotinamide (NAM) and alpha- d-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at product:substrate ratios thermodynamically forbidden in the absence of ATP. With ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases ( K m (NAM) from 855 to 5 nM), and the K eq shifts -2.1 kcal/mol toward NMN formation. ADP-ATP isotopic exchange experiments support the formation of a high-energy phosphorylated intermediate (phospho-H247) as the mechanism for altered catalytic efficiency during ATP hydrolysis. NAMPT captures only a small portion of the energy generated by ATP hydrolysis to shift the dynamic chemical equilibrium. Although the weak energetic coupling of ATP hydrolysis appears to be a nonoptimized enzymatic function, closer analysis of this remarkable protein reveals an enzyme designed to capture NAM with high efficiency at the expense of ATP hydrolysis. NMN is a rate-limiting precursor for recycling to the essential regulatory cofactor, nicotinamide adenine dinucleotide (NAD (+)). NMN synthesis by NAMPT is powerfully inhibited by both NAD (+) ( K i = 0.14 muM) and NADH ( K i = 0.22 muM), an apparent regulatory feedback mechanism.     

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.     

Transport of nicotinamide adenine dinucleotide in an unc mutant of Escherichia coli.

The presence and some properties of an NAD+ transport system were examined in PA5, a Mg, Ca-ATPase [EC 3.6.1.3]-defective mutant strain of Escherichia coli W2252. NAD+ uptake was stimulated by exogenous energy sources and dependent on external substrate concentrations with an apparent Km of about 25 micrometer. Most of the radioactivity from [14C]-NAD+ accumulated in the cells was identified as NAD+. [14C]NAD+ uptake was competively inhibited by unlabeled NAD+, NADP+, NMN+ or nicotinamide. Similar uptake activity was also observed in W2252.     

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.     

Nicotinamide phosphoribosyltransferase/visfatin does not catalyze nicotinamide mononucleotide formation in blood plasma

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined K(m) values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases K(m) values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of "NAMPT-mediated systemic NAD biosynthesis." Our study would advance current understanding of visfatin physiology.     

Studies of NAD kinase and NMN:ATP adenylyltransferase in Haemophilus influenzae

Previous studies of Haemophilus influenzae documented the importance of several pyridine nucleotide-dependent enzymes in processing extracellular NAD and NMN to satisfy the V-factor growth requirement of the organism. The substrate specificities of two of these enzymes. NMN:ATP adenylyltransferase and NAD kinase, were investigated following partial purification. The ability of the transferase to utilize 3-acetylpyridine mononucleotide and 3-aminopyridine mononucleotide as substrates for the synthesis of the corresponding dinucleotides was demonstrated. The NAD kinase was observed to accept 3-acetylpyridine adenine dinucleotide as a substrate but failed to utilize 3-aminopyridine adenine dinucleotide. The mononucleotides of 3-acetylpyridine and 3-aminopyridine were shown to be as effective as the corresponding dinucleotides in the support of growth and inhibition of growth of H. influenzae, respectively. Inhibition of growth of H. influenzae by submicromolar 3-aminopyridine adenine dinucleotide was shown to occur because 3-aminopyridine mononucleotide was produced from it in reactions catalysed by the H. influenzae periplasmic nucleotide pyrophosphatase. The presence of an additional important pyridine nucleotide-dependent enzyme, NMN glycohydrolase, is also reported.     

An enzymatic method for the measurement of nicotinamide mononucleotide pyrophosphorylase in cells and tissues

A simple enzymatic method is described for the measurement of NMN pyrophosphorylase in tissue homogenates at levels as low as 10(-12) to 10(-9) mol. The product, nicotinamide mononucleotide, is converted to NAD using NAD pyrophosphorylase and the NAD is quantified in an enzymatic cycling assay. The enzyme described here is stimulated more at low concentrations of Mn2+ than Mg2+. ATP is not required for NMN pyrophosphorylase activity; the reaction is neither stimulated nor inhibited by ATP concentrations as high as 3 mM. The enzyme is totally dependent on phosphoribosylpyrophosphate. The method is highly reproducible in all tissues examined. Various cell lines and tissues from mouse were analyzed for NMN pyrophosphorylase.     

Metabolic engineering of Escherichia coli for biosynthesis of β-nicotinamide mononucleotide from nicotinamide

The β-nicotinamide mononucleotide (NMN) is a key intermediate of an essential coenzyme for cellular redox reactions, NAD. Administration of NMN is reported to improve various symptoms, such as diabetes and age-related physiological decline. Thus, NMN is attracting much attention as a promising nutraceutical. Here, we engineered an Escherichia coli strain to produce NMN from cheap substrate nicotinamide (NAM) and glucose. The supply of in vivo precursor phosphoribosyl pyrophosphate (PRPP) and ATP was enhanced by strengthening the metabolic flux from glucose. A nicotinamide phosphoribosyltransferase with high activity was newly screened, which is the key enzyme for converting NAM to NMN with PRPP as cofactor. Notably, the E. coli endogenous protein YgcS, which function is primarily in the uptake of sugars, was firstly proven to be beneficial for NMN production in this study. Fine-tuning regulation of ygcS gene expression in the engineered E. coli strain increased NMN production. Combined with process optimization of whole-cell biocatalysts reaction, a final NMN titre of 496.2 mg l^-1 was obtained.     

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.     

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.     

Crystal structure of human nicotinamide riboside kinase

Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.     

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.