NAD(P) biosynthesis enzymes as potential targets for selective drug design.

NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed.

Synthesis and biological evaluation of NAD analogs as human pyridine nucleotide adenylyltransferase inhibitors.

NAD analogs modified at the ribose adenylyl moiety, named N-2'-MeAD and Na-2'-MeAD, were synthesized as ligands of pyridine nucleotide (NMN/NaMN) adenylyltransferase (NMNAT). Both dinucleotides resulted selective inhibitors against human NMNAT-3 isoenzyme.

Structure and function of nicotinamide mononucleotide adenylyltransferase.

The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily, catalyzes the reaction NMN + ATP = NAD + PPi, representing the final step in the biosynthesis of NAD, a molecule playing a fundamental role as a cofactor in cellular redox reactions. NAD also serves as the substrate for reactions involved in important regulatory roles, such as protein covalent modifications, like ADP-ribosylation reactions, as well as Sir2 histone deacetylase, a recently discovered class of enzymes involved in the regulation of gene silencing. This overview describes the most recent findings on NMNATs from bacteria, archaea, yeast, animal and human sources, with detailed consideration of their major kinetic, molecular and structural features. On this regard, the different characteristics exhibited by the enzyme from the various species are highlighted. The possibility that NMNAT may represent an interesting candidate as a target for the rational design of selective chemotherapeutic agents has been suggested.

Dinucleoside polyphosphate NAD analogs as potential NMN adenylyltransferase inhibitors. Synthesis and biological evaluation.

Two dinucleoside polyphosphate NAD analogs, P1-(adenosine-5')-P3-(nicotinamide riboside-5')triphosphate (Np3A, 1) and P1-(adenosine-5')-P4-(nicotinamide riboside-5')tetraphosphate (Np4A, 2), were synthesized and tested as inhibitors of both microbial and human recombinant NMN adenylyltransferase. Compounds 1 and 2 proved to be selective inhibitors of microbial enzymes.

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.

Structure of nicotinamide mononucleotide adenylyltransferase: a key enzyme in NAD(+) biosynthesis.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in fundamental processes in cell metabolism. The enzyme nicotinamide mononucleotide adenylyltransferase (NMN AT) plays a key role in NAD(+) biosynthesis, catalysing the condensation of nicotinamide mononucleotide and ATP, and yielding NAD(+) and pyrophosphate. Given its vital role in cell life, the enzyme represents a possible target for the development of new antibacterial agents. RESULTS: The structure of NMN AT from Methanococcus jannaschii in complex with ATP has been solved by X-ray crystallography at 2.0 A resolution, using a combination of single isomorphous replacement and density modification techniques. The structure reveals a hexamer with 32 point group symmetry composed of alpha/beta topology subunits. The catalytic site is located in a deep cleft on the surface of each subunit, where one ATP molecule and one Mg(2+) are observed. A strictly conserved HXGH motif (in single-letter amino acid code) is involved in ATP binding and recognition. CONCLUSIONS: The structure of NMN AT closely resembles that of phosphopantetheine adenylyltransferase. Remarkably, in spite of the fact that the two enzymes share the same fold and hexameric assembly, a striking difference in their quaternary structure is observed. Moreover, on the basis of structural similarity including the HXGH motif, we identify NMN AT as a novel member of the newly proposed superfamily of nucleotidyltransferase alpha/beta phosphodiesterases. Our structural data suggest that the catalytic mechanism does not rely on the direct involvement of any protein residues and is likely to be carried out through optimal positioning of substrates and transition-state stabilisation, as is proposed for other members of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily.

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.

Synechocystis sp. slr0787 protein is a novel bifunctional enzyme endowed with both nicotinamide mononucleotide adenylyltransferase and 'Nudix' hydrolase activities.

Synechocystis sp. slr0787 open reading frame encodes a 339 residue polypeptide with a predicted molecular mass of 38.5 kDa. Its deduced amino acid sequence shows extensive homology with known separate sequences of proteins from the thermophilic archaeon Methanococcus jannaschii. The N-terminal domain is highly homologous to the archaeal NMN adenylyltransferase, which catalyzes NAD synthesis from NMN and ATP. The C-terminal domain shares homology with the archaeal ADP-ribose pyrophosphatase, a member of the 'Nudix' hydrolase family. The slr0787 gene has been cloned into a T7-based vector for expression in Escherichia coli cells. The recombinant protein has been purified to homogeneity and demonstrated to possess both NMN adenylyltransferase and ADP-ribose pyrophosphatase activities. Both activities have been characterized and compared to their archaeal counterparts.

Glioma cells induce gamma-glutamyl transpeptidase activity in cultured blood but not lymphatic endothelial cells.

Glial cells have been shown to induce enzyme activities, which are characteristically high in brain capillaries, in cerebral and non cerebral endothelial cells in culture. We evaluated the activity of gamma-glutamyl transpeptidase (gamma GT) in freshly isolated (ex vivo) bovine blood (BEC) and lymphatic (LEC) endothelial cells. We also tested the effect of C6 glioma cell conditioned medium (GCM) on gamma GT activity of BEC and LEC in primary culture. Ex vivo BEC had a high gamma GT activity, only 9% of which was retained in culture. After exposure to GCM, however, gamma GT activity of cultured BEC was twofold higher than with control medium. By contrast gamma GT activity was extremely low in ex vivo LEC and did not significantly increase in cultured LEC exposed to GCM. These data show that the basal levels of gamma GT are markedly different in BEC and LEC and also that, unlike BEC, LEC are not capable of producing more gamma GT in response to glial stimulation.

Three-minute high-performance liquid chromatographic assay for NMN adenylyltransferase using a 20-mm-long reversed-phase column.

NMN adenylyltransferase (NAD pyrophosphorylase; NMNAT) reversibly catalyzes the synthesis of NAD from ATP and NMN. In this paper, we describe a rapid and sensitive high-performance liquid chromatographic assay for NMNAT, which uses a 20-mm-long C18 reversed-phase (RP) column. The activity was measured by separating in less than 3 min the substrates (NMN and ATP) from the product (NAD) with 0.1 M potassium phosphate, pH 6.0, at a 2 ml/min flow-rate and 22 degrees C. NAD was directly quantitated from its ultraviolet absorbance. Amounts of NAD as small as 25 pmol could be measured. The activity value closely agreed with that determined by the spectrophotometric assay. This method was successfully applied to the determination of NMNAT activity in human placental and bull testis extracts, as well as in rat pheochromocytoma (PC12) cells.

The antitumor drug, 1,3-bis(2-chloroethyl)-1-nitroso-urea, inactivates human nicotinamide mononucleotide adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) from human placenta is rapidly inactivated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). A similar inactivation is observed with other C- and N-nitroso compounds. The inactivation by BCNU is dependent on incubation time, temperature and BCNU concentration. Protective reagents for -SH groups, dithiothreitol and beta-mercaptoethanol, and the substrate NMN are very effective in protecting NMN adenylyltransferase from BCNU inactivation and in preserving its catalytic properties, while ATP is less efficient. Incubation of BCNU-inactivated and dialysed NMN adenylyltransferase with dithiothreitol results in a partial recovery of the enzymatic activity.

Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties.

A pyrimidine nucleotidase with unique specificity has been obtained for the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3'-monophosphates. Km values for 3'dUMP, 3'UMP, 5'dUMP, 5'UMP, 5-fluoro-2'dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphate, and the nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its activity, while heavy metals cations were strongly inhibitory. The enzyme activity was inhibited by metal chelating agents and it was sensitive to thioreactive reagents. The isoelectric point was 5.4 and the optimum activity pH was 6.5.

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.

ADPRibosylation of chicken red cell tubulin and inhibition of microtubule self-assembly in vitro by the NAD(+)-dependent avian ADPRibosyl transferase.

Chicken erythrocyte tubulin was found to undergo NAD(+)-dependent ADPribosylation in vitro in the presence of ADPRtransferase also isolated from avian red blood cells. Unlike the low level of ADPR incorporation catalyzed by Cholera and Pertussis toxins (i.e., less than 0.005 mol ADPR/mol tubulin), the avian system displayed a much higher stoichiometry of 0.8-1.2 mol ADPR/mol tubulin. Modification resulted in potent inhibition of microtubule self-assembly, even in the presence of bovine brain microtubule-associated proteins or with the addition of pre-assembled microtubules.

Microtubule-associated protein autophosphorylation alters in vitro microtubule dynamic instability.

While phosphorylation of high-molecular-weight microtubule-associated proteins (MAPs) alters the assembly properties of microtubules in vitro, virtually nothing is known about the influence of MAP phosphorylation on the time-scale of microtubule polymer length redistribution. The latter has been used as an index of microtubule assembly/disassembly turnover as predicted by the dynamic instability model (Mitchison, T.M. and Kirschner, M.W. (1984) Nature 312, 237-242). We have now determined that under conditions leading to the incorporation of 8-10 mol phosphoryl groups per mol MAP-2 (and about 0.2 mol phosphoryl groups per mol MAP-1 and tau), we can reproducibly observe significant acceleration in the polymer length redistribution process in a manner consistent with greater microtubule dynamic instability. We have also found that MAP phosphorylation resulted in more extensive release of MAPs from microtubules as a function of increasing salt concentration. These results are consistent with a weakening of MAP-microtubule interactions upon phosphorylation.

Evidence for an inhibitory effect exerted by yeast NMN adenylyltransferase on poly(ADP-ribose) polymerase activity.

We have previously reported for the first time the purification to homogeneity of the enzyme NMN adenylyltransferase (EC 2.7.7.1) from yeast and its major molecular and catalytic properties. The homogeneous enzyme was found to be a glycoprotein containing 2% carbohydrate and 1 mol of adenine residue and 2 mol of phosphate covalently bound per mole of protein. Such a stoichiometry, apparently consistent with that of ADP-ribose, prompted us to further investigate the possibility that NMN adenylyltransferase could be subjected to poly(ADP-ribosylation) in vitro in a reconstituted system. Poly(ADP-ribose) polymerase was purified to homogeneity from bull testis by means of a rapid procedure involving two batchwise steps on DNA-agarose and Reactive Blue 2 cross-linked agarose and a column affinity chromatography step on 3-aminobenzamide-Sepharose; the optimal conditions for the poly(ADP-ribosylation) of exogenous substrates were determined. When pure NMN adenylyltransferase was incubated in the presence of the homogeneous poly(ADP-ribose) polymerase, a marked inhibition of the polymerase was observed, both in the presence and in the absence of histones, while the activity of NMN adenylyltransferase was not affected. The inhibition could not be prevented by increasing the concentrations of either DNA or NAD. Mg2+ did not affect the activity or the inhibition. The significance of such a phenomenon is at present unknown, but it may be of biological relevance in view of the close topological and metabolic relationship between the two enzymes.

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.