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.

Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5'-nucleotidase.

Human erythrocyte pyrimidine 5'-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.

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.

Enzymology of NAD+ homeostasis in man.

This review describes the enzymes involved in human pyridine nucleotide metabolism starting with a detailed consideration of their major kinetic, molecular and structural properties. The presentation encompasses all the reactions starting from the de novo pyridine ring formation and leading to nicotinamide adenine dinucleotide (NAD(+)) synthesis and utilization. The regulation of NAD(+) homeostasis with respect to the physiological role played by the enzymes both utilizing NAD(+) through the nonredox NAD(+)-dependent reactions and catalyzing the recycling of the common product, nicotinamide, is discussed. The salient features of other enzymes such as NAD(+) pyrophosphatase, nicotinamide mononucleotide 5'-nucleotidase, nicotinamide riboside kinase and nicotinamide riboside phosphorylase, described under 'miscellaneous', are likewise presented.

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.

Phytotoxic protein PcF, purification, characterization, and cDNA sequencing of a novel hydroxyproline-containing factor secreted by the strawberry pathogen Phytophthora cactorum.

A novel protein factor, named PcF, has been isolated from the culture filtrate of Phytophthora cactorum strain P381 using a highly sensitive leaf necrosis bioassay with tomato seedlings. Isolated PcF protein alone induced leaf necrosis on its host strawberry plant. The primary structure and cDNA sequence of this novel phytotoxic protein was determined, and BLAST searches of Swiss-Prot, EMBL, and GenBank(TM)/EBI data banks showed that PcF shared no significant homology with other known sequences. The 52-residue PcF protein, which contains a 4-hydroxyproline residue along with three S-S bridges, exhibits a high content of acidic sidechains, accounting for its isoelectric point of 4.4. The molecular mass of isolated PcF is 5,622 +/- 0.5 Da as determined by mass spectrometry and matches that calculated from the deduced amino acid sequence with cDNA sequencing. The cDNA sequence indicates that PcF is first produced as a larger precursor, comprising an additional N-terminal, 21-residue secretory signal peptide. Maturation of this protein involves the hydroxylation of proline 49, a feature that is unique among other known secreted fungal phytopathogenic proteins.

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.

Human erythrocyte pyrimidine 5-nucleotidase, PN-I, is identical to p36, a protein associated to lupus inclusion formation in response to alpha-interferon.

Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an alpha-interferon-induced protein. PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library. PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme. The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36. Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I. Molecular properties of the two proteins were essentially the same. We conclude that p36 and PN-I are identical proteins. (Blood. 2000;96:1596-1598)

The Escherichia coli NadR regulator is endowed with nicotinamide mononucleotide adenylyltransferase activity.

The first identification and characterization of a catalytic activity associated with NadR protein is reported. A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases. The Escherichia coli nadR gene was cloned into a T7-based vector and overexpressed. In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.

Pyrimidine nucleotidases/phosphotransferases from human erythrocyte.

Two cytoplasmic pyrimidine 5'-nucleotidase have been purified from human erythrocytes to homogeneity and partially characterized. The two enzymes, indicated as PN-I and PN-II, preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates, respectively. The kinetic analysis demonstrate that pyrimidine 5'-nucleotidases, in the presence of suitable nucleoside substrates, can operate as phosphotransferases by transferring phosphate to various nucleoside acceptors, including nucleoside analogues known as important drugs widely used in chemotherapy.

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.

Identification of the archaeal NMN adenylytransferase gene.

Increasing evidence on the importance of fluctuations in NAD+ levels in the living cell is accumulating. Therefore a deeper knowledge on the regulation of coenzyme synthesis and recycling is required. In this context the study of NMN adenylyltransferase (EC 2.7.7.1),. a key enzyme in the NAD+ biosynthetic pathway, assumes a remarkable relevance. We have previously purified to homogeneity and characterized the protein from the thermophilic archaeon Sulfolobus solfataricus. The determination of partial sequence of the S. solfataricus enzyme, together with the recent availability of the genome sequence of the archaeon Methanococcus jannaschii, allowed us, based on sequence similarity, to identify the M. jannaschii NMN adenylyltransferase gene. As far as we know from literature, this is the first report on the NMN adenylyltransferase gene.

Enzymology of NAD+ synthesis.

Beyond its role as an essential coenzyme in numerous oxidoreductase reactions as well as respiration, there is growing recognition that NAD+ fulfills many other vital regulatory functions both as a substrate and as an allosteric effector. This review describes the enzymes involved in pyridine nucleotide metabolism, starting with a detailed consideration of the anaerobic and aerobic pathways leading to quinolinate, a key precursor of NAD+. Conversion of quinolinate and 5'-phosphoribosyl-1'-pyrophosphate to NAD+ and diphosphate by phosphoribosyltransferase is then explored before proceeding to a discussion the molecular and kinetic properties of NMN adenylytransferase. The salient features of NAD+ synthetase as well as NAD+ kinase are likewise presented. The remainder of the review encompasses the metabolic steps devoted to (a) the salvaging of various niacin derivatives, including the roles played by NAD+ and NADH pyrophosphatases, nicotinamide deamidase, and NMN deamidase, and (b) utilization of niacins by nicotinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase.

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.