Chemoenzymatic Preparation of 4'-Thioribose NAD.

This chemoenzymatic procedure describes a strategy for the preparation of 4'-thioribose nicotinamide adenine dinucleotide (S-NAD+ ), including chemical synthesis of nicotinamide 4'-riboside (S-NR), recombinant expression and purification of two NAD+ biosynthesis enzymes nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), and enzymatic synthesis of S-NAD+ . The first basic protocol describes the procedures for introduction of nicotinamide onto 4'-thioribose and subsequent deprotection to generate S-NR as the key intermediate for enzymatically synthesizing S-NAD+ . In the second basic protocol, experimental methods are detailed for the production of recombinant human NRK1 and NMNAT1 to catalyze conversion of S-NR to S-NAD+ . The third basic protocol presents the enzymatic approach for the generation of S-NAD+ from S-NR precursor. © 2019 by John Wiley & Sons, Inc.

Characterization and application of a novel nicotinamide mononucleotide adenylyltransferase from Thermus thermophilus HB8.

Herein, we describe a novel enzymatic cycling method to measure nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), which are precursors of NAD biosynthesis. A gene encoding an NMN adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified in Thermus thermophilus HB8. The gene from T. thermophilus (TtNMNAT) was engineered for expression in Escherichia coli and the recombinant enzyme found to be stable, retaining full activity after incubation for 45 min at 70°C. The Km values for NMN and ATP were calculated to be 0.263 and 1.27 mM, respectively, with a Vmax value of 60.3 µmoL/min/mg. TtNMNAT was successfully applied to the colorimetric NMN or NaMN assays, which employed (i) adenylation of NMN to NAD by TtNMNAT or adenylation of NaMN to deamido-NAD (NaAD) by TtNMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 6.3.1.5) and (ii) an NAD cycling reaction using 12α-hydroxysteroid dehydrogenase (12α-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced WST-8. This enzymatic cycling method enabled detection of 0.5 µM (12.2 nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer.

Nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi (TcNMNAT): a cytosol protein target for serine kinases

Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite’s NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.

Nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi (TcNMNAT): a cytosol protein target for serine kinases.

Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite's NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.

Regulation of inflammatory cytokine expression in pulmonary epithelial cells by pre-B-cell colony-enhancing factor via a nonenzymatic and AP-1-dependent mechanism.

Although our previous studies found Pre-B-cell colony-enhancing factor (PBEF) as a highly up-regulated gene in acute lung injury that could stimulate expressions of other inflammatory cytokines, the underlying molecular mechanisms remain to be fully elucidated. Growing evidence indicates that PBEF is a nicotinamide phosphoribosyltransferase involved in the mammalian salvage pathway of NAD synthesis. This study was designed to determine whether the effect of PBEF to stimulate expressions of inflammatory cytokines depends on its enzymatic activity. We prepared two human PBEF mutant (H247E and H247A) recombinant proteins and overexpressing constructs for their overexpressions in A549 cells and confirmed that enzymatic activities of both mutants were nearly or completely abolished. Two mutants stimulated interleukin-8 (IL-8) expression at both the mRNA level and protein level just as equally effective as the wild-type PBEF did. These effects were due to the increased transcription, not the mRNA stability, of the IL-8 gene. Reporter gene assays and gel shift experiments indicated that AP-1 transcription factor is required to mediate these effects. SB203580, a p38 MAPK pathway inhibitor, and JNK inhibitor 1 can attenuate these effects. Both PBEF mutants similarly stimulated the expression of two other inflammatory cytokines: IL-16 and CCR3. These results indicate that PBEF stimulated expression of IL-8, IL-16, and CCR3 via its non-enzymatic activity. This effect is AP-1-dependent, in part via the p38 MAPK pathway and the JNK pathway. This finding reveals a new insight, which may manifest a novel role of PBEF in the pathogenesis of acute lung injury and other inflammatory disorders.

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.

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis.

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.

Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase.

A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases. Northern blotting detected a major 3.1-kilobase mRNA transcript as well as a minor 4.1-kilobase transcript in all human tissues examined. In several cancer cell lines, lower levels of mRNA expression were clearly evident. The gene encoding the human enzyme was mapped to chromosome band 1p32-35. High efficiency bacterial expression yielded 1.5 mg of recombinant enzyme/liter of culture medium. The molecular and kinetic properties of recombinant human NMN adenylyltransferase provide new directions for investigating metabolic pathways involving this enzyme.

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.

Characterization of nicotinamide mononucleotide adenylyltransferase from thermophilic archaea.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the synthesis of NAD+ and nicotinic acid adenine dinucleotide. It has been purified to homogeneity from cellular extracts of the thermophilic archaeon Sulfolobus solfataricus. Through a database search, a highly significant match was found between its N-terminal sequence and a hypothetical protein coded by the thermophilic archaeon Methanococcus jannaschii MJ0541 open reading frame (GenBank accession no. U67503). The MJ0541 gene was isolated, cloned into a T7-based vector, and expressed in Escherichia coli cells, yielding a high level of thermophilic NMN adenylyltransferase activity. The expressed protein was purified to homogeneity by a single-step chromatographic procedure. Both the subunit molecular mass and the N-terminal sequence of the pure recombinant protein were as expected from the deduced amino acid sequence of the MJ0541 open reading frame-encoded protein. Molecular and kinetic properties of the enzymes from both archaea are reported and compared with those already known for the mesophilic eukaryotic NMN adenylyltransferase.

NMN adenylyltransferase from bull testis: purification and properties.

The purification procedure of NMN adenylyltransferase from bull testis presented here consists of a heat step and an acidic precipitation followed by four chromatographic steps, including dye ligand, adsorption and hydrophobic chromatography. The final enzyme preparation subjected to non-denaturing and denaturating PAGE with silver nitrate staining exhibited a single band. At this step the enzyme appeared to be homogeneous. The M(r) value of the native enzyme calculated by gel filtration was about 133,000. The protein appeared to possess a quaternary structure with four subunits of apparent M(r) 33,000 without disulphide interchain bonds. Isoelectric experiments gave a pI of 6.2, and pH studies showed the possible presence of an acidic group in the active site having a pKa of 4.9. Analysis of the amino acid composition showed the presence of more acidic residues than basic ones, according to the pI value calculated by Mono P FPLC. The Ea calculated by Arrhenius plot gave an apparent value of 55.7 kJ/mol. The Km values for NMN, ATP, NAD+ and PPi were 0.11, 0.023, 0.37 and 0.16 nM respectively. The polyclonal antiserum produced against the NMN adenylyltransferase reacted with the purified enzyme at different dilutions and recognized the enzyme in the homogenate as well.

Pyridine dinucleotide biosynthesis in archaebacteria: presence of NMN adenylyltransferase in Sulfolobus solfataricus.

The enzyme NMN adenylyltransferase, leading to NAD synthesis, has been observed for the first time in soluble extracts from the extreme acidothermophilic archaeon Sulfolobus solfataricus. Comparison of its molecular and kinetic properties with those of the enzyme isolated from prokaryotes and eukaryotes revealed significant differences, knowledge of which may contribute to the understanding of metabolic evolutionary mechanisms. The thermophilic enzyme shows a molecular mass of about 66,000 and an isoelectric point of 5.4. The Km values for ATP, NMN and nicotinic acid mononucleotide are 0.08 microM, 1.4 microM and 17 microM, respectively. The enzyme shows a remarkable degree of thermophilicity, with an activation energy of 95 kJ/mol.

NAD biosynthesis in human placenta: purification and characterization of homogeneous NMN adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from human placenta. The purification procedure consists of several chromatographic steps, including dye-ligand, adsorption, and hydrophobic interaction chromatography. The final enzyme preparation is homogeneous as judged by a single silver stainable band on both nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 132,000, as determined by gel filtration on a Superose 12 HR 10/30 fast protein liquid chromatography column. The protein possesses a quaternary structure and is composed of four apparently identical M(r) 33,000 subunits. Isoelectrofocusing experiments give multiple pI values ranging from pH 4.7 to 6.6. Optimum pH study shows a plateau extending from pH 6.0 to pH 9.0. Km values for NMN, ATP, NAD+, and PPi are 38, 23, 67, and 125 microM, respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and beta-NMNH were found to significantly inhibit the enzyme activity.

Recent observations on the structure and the properties of yeast NMN adenylyltransferase.

A homogeneous preparation of yeast NMN adenylyltransferase (EC 2.7.7.1) showed microheterogeneity, which was revealed by FPLC (Fast Protein Liquid Chromatography) ion exchange chromatography. The resolved components have been characterized with respect to electrophoretic behavior and adenine content. The results led to a hypothesis about a possible role of poly(ADP-ribosylation) in modulating the enzyme activity.

NAD pyrophosphorylase from yeast chromatin. Purification and properties.

NAD pyrophosporylase has been purified to homogeneity from baker's yeast. The purification procedure is relatively simple and consists of high salt extraction of enzyme activity, precipitation with polyethylenimine followed by ion exchange and by ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue-stainable band when run on non-denaturing and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200,000 calculated by gel filtration and sucrose gradient centrifugation. The protein possess quaternary structure and is composed by four apparently identical Mr 50,000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. Isoelectric point is 6.2. Amino acid composition shows the presence of 28 half-cystine residues, in agreement with the results obtained by titrating the enzyme in denaturing conditions with Ellman's reagent upon previous incubation with sodium borohydride. NAD pyrophosphorylase is a glycoprotein containing 2% sugar, 2 moles of alkali-labile phosphate per enzyme mol, and 1 mol of adenine moiety per enzyme mol. Therefore the possibility that the enzyme is ADP-ribosylated exists. Km for ATP, NMN and NaMN are 0.11 mM, 0.19 mM and 5 mM respectively. Kinetic analysis reveals a behaviour which is consistent with an ordered sequential Bi-Bi mechanism. pH optimum is in the range 7.2-8.4.

Nicotinamide mononucleotide adenylyltransferase. Molecular and enzymatic properties of the homogeneous enzyme from baker's yeast.

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation. The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. The isoelectric point is 6.2. Amino acid composition analysis shows the presence of 28 half-cystine residues. The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride. NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme. Therefore, the possibility that the enzyme is ADP-ribosylated exists. The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively. Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism. The pH optimum is in the range 7.2-8.4.