NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Mobley HLT, Mendz GL, Hazell SL, editors. Helicobacter pylori: Physiology and Genetics. Washington (DC): ASM Press; 2001.

Cover of Helicobacter pylori

Helicobacter pylori: Physiology and Genetics.

Show details

Chapter 13Nucleotide Metabolism


School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney, NSW, 2052, Australia

Nucleotides have a central role in the physiology of organisms as building blocks of nucleic acids, storage of chemical energy, carriers of activated metabolites for biosynthesis, structural moieties of coenzymes, and metabolic regulators. A complete understanding of the nucleotide metabolism of Helicobacter pylori is of fundamental interest to microbiology and also will help in the development of new anti-H. pylori therapies. Owing to the complex and varied interactions of nucleotides present in normal functions, cells have an essential need to maintain tightly regulated pools of these compounds, which will serve to keep nucleotide balance and avoid wasting resources on end products not required by the organism.

Pyrimidines and purines are essential for the synthesis of nucleoside triphosphates, which are precursors of nucleic acids. Nucleoside polyphosphates are formed by successive phosphorylations of their mono-phosphate counterparts. 5-Phospho-α-d-ribosyl-l-pyrophosphate (PRPP) is synthesized from ATP and ribose 5-phosphate by the action of phosphoribosyl pyrophosphate synthetase. This enzyme is encoded by gene HP0742 (prsA) in H. pylori. The ribose 5-phosphate moiety of nucleotides is derived from PRPP in de novo synthesis and in some salvage pathways. Ribonucleoside monophosphates are precursors of deoxyribonucleoside monophosphates and may be synthesized de novo from simple precursors or formed via salvage pathways.

Pyrimidine Ribonucleotides

Pyrimidine ribonucleotide synthesis in H. pylori was investigated by examining the incorporation of pyrimidine ring precursors and preformed pyrimidines and the activities of enzymes involved in their biosynthetic pathways (17).

De Novo Pyrimidine Nucleotide Synthesis

The pathway responsible for the de novo synthesis of UTP and CTP, two precursors of RNA, comprises nine enzymes (Fig. 1). The ability of H. pylori to grow in a defined medium without preformed pyrimidines was studied by successive passage experiments, and the results indicate that the bacterium can grow and replicate by relying exclusively on the de novo biosynthesis of pyrimidine nucleotides (19, 31). The enzyme activities of the de novo pyrimidine synthesis pathway of H. pylori have been identified in situ, and putative genes coding for the corresponding enzymes have been found in its genome (3, 33).

Figure 1

Figure 1

. De novo pyrimidine biosynthesis pathway. The enzymes encoded by the pyr genes are pyrA, carbamoyl phosphate synthase; pyrB, aspartate carbamoyl transferase; pyrC, dihydroorotase; pyrD, dihydroorotase dehydrogenase; pyrE, orotate phosphoribosyltransferase; (more...)

Carbamoyl phosphate synthetase

Carbamoyl phosphate is a substrate for the first reaction of the pathway and is formed by a synthetase that catalyzes the reaction from glutamine or ammonium, bicarbonate, and ATP. Carbamoyl phosphate forms a metabolic branchpoint for the arginine and the de novo pyrimidine pathways. The incorporation of radioactive carbon atoms from bicarbonate into nucleic acids suggests the presence of a de novo pyrimidine nucleotide synthesis pathway in H. pylori (17), although it is possible that this incorporation may take place also via de novo purine biosynthesis. Analysis of the H. pylori DNA sequence identified the genes HP1237 and HP0919, sharing similarities with the pyrAa gene from Salmonella enterica serovar Cholerasuis and the pyrAb gene from Bacillus caldolyticus (3, 33), which encode carbamoyl phosphate synthetases; these findings support the interpretation of the experimental data.

Aspartate carbamoyl transferase

In the first reaction committed solely to pyrimidine biosynthesis, carbamoyl phosphate is converted to carbamoyl aspartate by its condensation with the amino group of aspartate in a reaction catalyzed by aspartate carbamoyl transferase (ACTase). In Escherichia coli, this enzyme is a dodecamer composed of two catalytic trimers (c3) and three regulatory dimers (r2). The gene for the catalytic chain, pyrB, and the gene for the regulatory chain, pyrI, are organized in an operon transcribed from B to I (21). The gene HP1084 in the H. pylori genome is similar to the pyrB gene from Bacillus subtilis, but no gene orthologous to pyrI seems to be present. The activity of this enzyme was measured in H. pylori lysates by radioactive tracer analysis (17). The kinetic and regulatory properties of H. pylori ACTase were investigated employing nuclear magnetic resonance spectroscopy, radioactive tracer analysis, and microtiter colorimetric assays (6). The apparent Kms were 0.6 and 11.6 mM for carbamoyl phosphate and aspartate, respectively, and there is no evidence of substrate inhibition at higher concentrations of either substrate. Optimal pH and temperature are 8.0 and 45 °C, respectively. Activity is observed with the l-but not the d-isomer of aspartate. Succinate and maleate inhibit enzyme activity competitively with respect to aspartate. The carbamoyl phosphate analogs acetyl phosphate and phosphonoacetic acid inhibit activity in a competitive manner with respect to carbamoyl phosphate. With limiting carbamoyl phosphate, purine and pyrimidine nucleotides, tripolyphosphate, pyrophosphate, and orthophosphate inhibit the enzyme competitively at millimolar concentrations. Ribose and ribose-5-phosphate at 10 mM concentration show 20 and 35% inhibition of enzyme activity, respectively. N-Phosphonoacetyl-l-aspartate (PALA) is the most potent inhibitor studied, with 50% inhibition of enzyme activity observed at 0.1 μM concentration. Inhibition by PALA is competitive with carbamoyl phosphate (Ki = 0.245 μM), and noncompetitive with aspartate (6, 7).

On the basis of catalytic and regulatory properties, ACTase can be grouped into three different classes: class A ACTase exhibits inhibition by all nucleotides, class B enzymes show inhibition by CTP and activation by ATP, and class C ACTase is unaffected by all nucleotides. ACTase is also usually a major point of regulatory control of the de novo biosynthetic pathway in bacteria, and the importance of ACTase in the overall survival of cells is well illustrated by the fact that inhibitors of this enzyme have been employed as antiproliferative drugs in both prokaryotes and eukaryotes (12, 32). Several features of the H. pylori enzyme activity differ from those of other ACTases, particularly from the well-characterized E. coli enzyme. The H. pylori ACTase substrate saturation curves display hyperbolic kinetics, but the E. coli curves exhibit sigmoidal characteristics (5). Sigmoidal substrate kinetics are also found for Streptococcus faecalis (9) and Paracoccus abyssi (26). Substrate inhibition at higher levels of aspartate is observed in both E. coli and S. faecalis (9); however, no such effect is seen in the study with H. pylori ACTase (6). The substrate kinetics of enzyme activity in this study is similar to that of Pseudomonas fluorescens, whose ACTase also lacks sigmoidal kinetics and substrate inhibition (13). The differences in kinetics and nucleotide regulation form the criteria for the classification of ACTase; based on the kinetic and regulatory features of enzyme activity, the ACTase of H. pylori can be grouped as a class A enzyme, although some specific features of its regulation appear unique (6). Several catalytic properties differ from other known class A enzymes and distinguish the H. pylori ACTase activity. It was found that the hyperbolic carbamoyl phosphate saturation curve of the P. fluorescens class A ACTase becomes sigmoidal upon the addition of CTP or UTP (1, 22), but the results obtained for H. pylori indicate no deviation from the hyperbolic kinetics in the presence of these nucleotides. Another difference mentioned earlier is the effects of substrate analogs; a class A enzyme from Vibrio costicola shows slight activation in the presence of succinate at low aspartate concentration (2), whereas the H. pylori enzyme shows no such activation. Thus, some characteristics of the substrate saturation curves described for H. pylori ACTase differ from those common to this group of ACTase. The results of this investigation suggest that the extent of regulation of ACTase activity, and thus de novo pyrimidine biosynthesis, depends on the concentrations and availability to H. pylori cells of certain metabolic regulators in their natural environment, and that the first committed step in the de novo biosynthesis pathway in cell-free extracts of H. pylori is highly regulated.

To investigate the role of ACTase in the survival of H. pylori, an attempt was made to construct defined isogenic pyrB mutants deficient in this enzyme activity by insertional mutagenesis of the gene encoding for chloramphenicol resistance (8). To facilitate the construction of a pyrB-disrupted copy in E. coli, the complete structural gene for ACTase from H. pylori strain RU1 was cloned into E. coli by complementation of a pyrB auxotrophic mutant. The H. pylori pyrB gene is 924 bp and encodes a theoretical protein with an Mr of 34,278, in agreement with the pyrB of H. pylori strains 26695 (33) and J99 (3). The predicted protein size was very similar to that of the catalytic chain of other bacterial (15, 16) and eukaryotic ACTase (13), supporting the proposal that the 34-kDa species is a universal occurrence in these enzymes (4). The H. pylori gene has high similarity to other bacterial pyrB genes, and the phylogenetic clustering with different species is consistent with functional characteristics of the ACTase. Analysis of the region upstream of the pyrB gene does not reveal sequences that conform to the consensus recognition site associated with attenuation control, also suggesting that the H. pylori gene does not appear to be regulated by mechanisms modulated by pyrimidine limitation described for E. coli and Bacillus spp. (27, 29, 35). The finding that levels of recombinant ACTase activity are unaffected by the addition of uracil further suggests that regulation at the level of enzyme synthesis of the pyrB-encoded protein may not occur in H. pylori by known mechanisms, similar to the finding in several Pseudomonas spp. (10). The transcription initiation site for H. pylori pyrB-mRNA was mapped by a protocol based on ligation-anchored PCR, and potential promoter regions were identified. In multiple transformations of H. pylori cells, no chloramphenicol-resistant pyrB mutants were isolated. Successful mutagenesis of other H. pylori genes and PCR amplification of the recombined gene demonstrated that the ACTase-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the pyrB gene with the chloramphenicol-pyrB-disrupted copy. This work extended the understanding of pyrB beyond the analysis of primary structure data (3, 33) and suggested that the ACTase enzyme is essential for the survival of H. pylori (8).

From a biomedical perspective, the essential nature of H. pylori ACTase demonstrated the potential for targeting the pyrimidine pathway for therapeutic intervention. If the unique characteristics of the H. pylori enzyme could be exploited, it may allow for the rational design of agents that could irreversibly disrupt this biosynthetic pathway in the bacterium. Although a previous investigation showed that some drugs that target ACTase can be detoxified by H. pylori (7), there is great potential to alter selectively the nature of such compounds or to use them in combination.

Dihydroorotase and dihydroorotate dehydrogenase

Orotate is formed by the action of dihydroorotase (DHOase) and dihydroorotate dehydrogenase (DHODHase). The genes HP0266 and HP0581 are orthologous to the gene pyrC, which encodes the DHOase of Pseudomonas putida, and the gene HP1011 is homologous to pyrD, which encodes the DHODHase of Haemophilus influenzae. The activity of H. pylori DHOase has been measured in situ employing 1H-nuclear magnetic resonance (NMR) spectroscopy (31). This enzyme activity is inhibited by primaquine, lawsone, and juglone, which are known inhibitors of DHOase in other organisms, and by oxantel, an inhibitor of H. pylori fumarate reductase (19, 31). The effects of these compounds on the growth and viability of cells were measured in solid and liquid cultures, and they have bactericidal effects at various concentrations (19, 31). The lethal effects on cells of blockage of this pathway agree with the conclusion that the de novo biosynthesis of pyrimidine nucleotides is essential for growth and viability of the bacterium. Recently, H. pylori DHODHase has been expressed and characterized in E. coli mutants lacking pyrD (11). The enzyme is a family 2 DHODHase like the one found in other gram-negative bacteria and from mammalian sources. It is a membrane-associated flavoprotein that utilizes an endogenous FMN redox cofactor and exogenous coenzyme Q6 as an electron acceptor, although it can donate electrons also to coenzyme Q0, menaquinone, and menadione. Some pyrazole-based compounds are strong inhibitors of the H. pylori enzyme but not of the human enzyme (11). These compounds also have potent antibacterial effects against H. pylori cells but are not active against other gram-negative, gram-positive, or human cells (11), suggesting that they are selective antibacterial agents for H. pylori and confirming the finding that the enzymes of the de novo pyrimidine biosynthesis pathway are potential therapeutic targets (19).

Orotate phosphoribosyl transferase, orotidine-5′-phosphate decarboxylase

Orotate phosphoribosyl transferase (OPRTase) catalyzes the transfer of a ribose 5-phosphate moiety from PRPP to orotic acid, yielding the nucleotide orotidylic acid, which is subsequently decarboxylated to UMP by the action of the orotidine 5′-phosphate decarboxylase (ODCase). H. pylori has the coding capacity for these two enzymes whose activities in cells and lysates have been measured employing radio-tracer analysis and 31P-NMR spectroscopy (17). In the genome of the bacterium the gene HP1257 is homologous to the pyrE gene encoding OPRTase of Thermus aquaticus, and the gene HP0005 is homologous to the pyrF gene coding for ODCase of B. subtilis (3, 33).

A salient feature of the data on the de novo pyrimidine synthesis enzymes is the high activity measured for H. pylori ODCase. Although OPRTase and ODCase are different bacterial enzymes, in some microorganisms, such as certain yeast strains (36) and Serratia marcescens (39), they appear to form multi-functional enzyme complexes, which would channel or regulate the catalytic efficiency of the pathway. Evidence for such channeling of the OPRTase-ODCase complex has been obtained for a number of eukaryotic systems including Ehrlich ascites cells (34) and the protozoan parasite Crithidia luciliae (25). Thus, the high ODCase activity observed for H. pylori may indicate the presence of a multienzyme complex with a similar function.

UMP kinase, nucleoside diphosphokinase, and CTP synthase

The last steps in the generation of UTP involve phosphorylation of UMP to UDP and of UDP to UTP by the sequential action of UMP kinase and nucleoside diphosphokinase, two enzymes whose activities have been observed in H. pylori (17). The genes HP0777 and HP0198 are orthologs of the genes pyrH and ndk, encoding UMP kinase and nucleoside diphosphokinase, respectively (3, 33).

Finally, amination of UTP by CTP synthase converts it to CTP. This enzyme activity was measured in bacterial lysates employing radiolabeled UTP (17), and gene HP0349 is homologous to the pyrG gene encoding CTP synthase in H. influenzae (3, 33).

Salvage of Preformed Pyrimidines

Salvage pathways enable cells to scavenge preformed nucleic bases and nucleosides for nucleotide synthesis or to reutilize bases and nucleosides produced endogenously as a result of nucleotide turnover, allowing circumvention of de novo pathways.

Utilization of preformed pyrimidine bases is limited in H. pylori. Radioactive tracer analysis experiments with orotate, an intermediate of the de novo pyrimidine nucleotide synthesis pathway, show that this pyrimidine is taken up by H. pylori cultures at moderate rates (17). Labeled precursors of pyrimidine salvage pathways such as uracil and uridine are significantly less well incorporated, although their uptake is linear at the beginning of the incubations (17). The linearity of the uptake of orotate, uracil, and uridine during the first 10 h of the time courses with cells suggests that these pyrimidine moieties were utilized intact. It is worth noticing that no gene similar to uraA, which encodes for a cytoplasmic membrane protein required for uracil uptake in E. coli, was found in the H. pylori genome (3, 33). Phosphoribosyltransferases are important for the salvage of free nucleobases for biosynthesis; they act by transferring the ribosyl-phosphate moiety of PRPP to a nucleotide base, yielding the corresponding nucleoside monophosphate (NMP). Enzyme activities for both orotate (OPRTase) and uracil (UPRTase) are observed in situ in H. pylori (17). In E. coli, the upp gene coding for UPRTase is the first gene of a bicistronic operon with uraA as the second gene. In H. pylori, the gene HP1257 with similarity to pyrE coding for T. aquaticus OPRTase (part of the de novo pathway described above) was identified, but no gene orthologous to upp was found. The gene HP1180 has similarity with nupC coding for a pyrimidine nucleoside transport protein in B. subtilis; however, H. pylori incorporates only very small quantities of uridine (17). No other genes orthologous to those encoding enzymes required for pyrimidine salvage in E. coli, such as udp and udk coding for uridine phosphorylase and uridine kinase, respectively, have been identified in H. pylori DNA (3, 33).

The metabolism of pyrimidine bases and nucleosides is well understood in many gram-negative bacteria. An important characteristic of E. coli and S. enterica serovar Typhimurium is that exogenous pyrimidine bases, nucleosides, and deoxynucleosides can be incorporated into nucleic acids. Other gram-negative bacteria such as Aerobacter aerogenes, Citrobacter freundii, Halobacterium cutiburum, Proteus vulgaris, Pseudomonas aeruginosa, Rhodopseudomonas sphaeroides (24), and S. marcescens (38) are able to metabolize exogenous precursors of pyrimidine nucleotides. In contrast, Neisseria meningiditis has a more restricted metabolism of bases and nucleosides; it can synthesize pyrimidine nucleotides by the de novo pathway, but uracil is the only pyrimidine nucleotide precursor that it is capable of incorporating (14).

In summary, much larger rates of utilization were measured for aspartate, bicarbonate, and orotate than for uracil, cytosine, thymine, or any of the pyrimidine nucleosides tested (17). The incorporation of uracil and uridine and the observation of UPRTase activity indicated that the bacterium is capable of obtaining pyrimidine nucleotides by salvage of precursors. However, in comparison to other gram-negative bacteria, H. pylori showed a limited ability to incorporate preformed bases and possibly nucleosides, suggesting a pyrimidine metabolism that may be similar to that of Neisseria spp.

Purine Nucleotides

The de novo synthesis of purine nucleotides is carried out by pathways that are similar throughout the biological world, but many organisms obtain their nucleotide needs utilizing preformed purine compounds through salvage pathways that take up available purine nucleobases and nucleosides.

Little is known about purine nucleotide biosynthesis in H. pylori, although studies have been carried out on the ability of the bacterium to survive by synthesizing purines de novo, and on its incorporation of preformed purine bases and nucleosides and the activities of enzymes involved in purine salvage pathways.

De Novo Purine Nucleotide Biosynthesis

A study of the basic nutrients required by H. pylori found that in defined media comprising a mixture of amino acids, salts, and vitamins, the lack of adenine or other preformed purines in the media reduced bacterial growth by 50%; and the bacterium grew better without the presence of uracil, guanine, and xanthine (28). An interpretation of these findings is that the bacterium requires salvage of purine nucleotide precursors for growth and proliferation.

Inosinate (IMP) is the purine ribonucleotide formed de novo in the pathway. IMP is formed in 10 enzymatic reactions by stepwise additions of functional groups to PRPP (Fig. 2). In the H. pylori genome, only the genes HP1218 and HP1112 have similarities with genes coding for enzymes required for IMP synthesis (3, 33). The first one is orthologous to purD, encoding glycinamide ribonucleotide synthetase, and the second one to purB, encoding the bifunctional enzyme adenylosuccinate lyase, which also has a function in the synthesis of AMP from IMP. These data from genome analyses agree with the initial findings suggesting that adenine was required for survival of H. pylori.

Figure 2

Figure 2

. De novo purine biosynthesis pathway. The enzymes encoded by the pur genes are purF, glutamine:PRPP-amido-transferase; purD, phosphoribosylglycinamide (GAR) synthetase; purN, GAR transformylase N; purT, GAR transformylase T; purL, phosphoribosyl-N-formylglycinamide (more...)

Although eight of the enzymes involved in de novo purine synthesis have not been identified in the genome of H. pylori, a study carried out with laboratory-adapted strains and low-passage isolates demonstrated full growth and viability of the bacterium after multiple passages in chemically defined media lacking purine bases, nucleosides, and nucleotides (20). The sustained growth and proliferation of the bacterium for five passages in defined media free of preformed purines in the presence of bovine serum albumin and catalase indicate that growth is not achieved by uptake of residual preformed purines that may have been carried into the media with bacteria harvested from plates or made available by lysis of a fraction of the cell populations. These results indicate that H. pylori is able to synthesize purines de novo to meet its purine nucleotide requirements.

To understand the role of adenine in the growth of H. pylori, cells were grown in chemically defined media with or without adenine under different conditions. The viability of bacterial cells decreases with each passage if both catalase and adenine are absent from the media but remains unchanged if exogenous catalase is present. In the absence of blood or serum, culture media for growing H. pylori are supplemented with catalase to reduce the concentrations of reactive oxygen species. It was confirmed that adenine supports H. pylori viability in the absence of catalase by adding adenine to the cultures and reversing the decline in the number of colony-forming units (20). Adenine is a scavenger of hydroxyl radicals (OH·), and cell growth experiments with this purine base and several other scavengers of OH· suggest that one of the roles of adenine may be the protection of cells against hydroxyl radicals (20).

Methotrexate (MTX) is a folate analog that inhibits dihydrofolate reductase and arrests growth of folate-requiring bacteria (30). Dose-response curves show strong bactericidal effects of MTX on H. pylori (20). Inhibition of the production of tetrahydrofolate may interfere with any of the biosynthetic reactions in which it functions as a one-carbon unit carrier, namely glycine and methionine biosynthesis, thymidylate synthesis, and purine biosynthesis. The nature of the inhibition of cell growth by MTX and the role of the purine de novo biosynthetic pathway in the survival of H. pylori were investigated by growing the bacterium in the presence of MTX and the bases adenine or hypoxanthine, or aminoimidazole carboxamide (20). The presence of the purine metabolites reverses the inhibition of growth produced by MTX, suggesting that the bactericidal effects of the antifolate on H. pylori occur by blockading purine de novo biosynthesis.

Although no gene ortholog of purF encoding glutamine:PRPP-amidotransferase, the first committed step in de novo purine biosynthesis (Fig. 2), has been identified in the H. pylori genome (3, 33), the presence of this enzyme activity in the bacterium has been demonstrated employing 1H- and 31P-NMR spectroscopy (Mendz, unpublished results). H. pylori lysates incubated with the substrates glutamine and PRPP yield as products glutamate and PPi (the third product of this enzymic reaction, 5-phosphoribosylamine, decomposes rapidly), indicating the presence of this amidotransferase activity in the bacterium. Azaserine and 6-diazo-5-oxonorleucine are structural analogs of glutamine and inhibit irreversibly glutamine amidotransferases in many organisms. The inhibition of glutamine:PRPP-amidotransferase activity in H. pylori lysates by these compounds provided evidence supporting the presence of this enzyme in the bacterium (Mendz, unpublished results).

The conversion of IMP to GTP or ATP requires four enzymes for each branch of the pathway, and the genes coding for these enzymes seem to be present in H. pylori DNA (Fig. 3). Involved in GDP synthesis are IMP dehydrogenase encoded by guaB orthologous to HP0829, GMP synthetase encoded by guaA orthologous to HP0409, and 5′-guanylate kinase encoded by gmk orthologous to HP0321. The enzymes for ADP synthesis are adenylosuccinate synthetase encoded by purA orthologous to HP0255, adenylosuccinate lyase encoded by purB orthologous to HP1112, and adenylate kinase encoded by adk orthologous to HP0618 (3, 33). GDP and most nucleoside diphosphates are converted to triphosphates through the action of nucleoside diphosphate kinase with ATP as the phosphate donor. The gene HP0198 found in the H. pylori genome has high similarity to the ndk gene encoding this enzyme in Myxococcus xanthus (3, 33). The presence of the genes of both pathways suggests that these are the routes utilized by the bacterium to synthesize ATP and GTP from inosinic acid. However, no guanylate kinase activity is observed in bacterial lysates, and H. pylori adenylate kinase is able to phosphorylate AMP with GTP or ITP as phosphate donors with production of GDP and IDP, respectively (18). These results suggest that the syntheses of GTP and ITP may follow alternative pathways and raise the possibility that the guaB and guaA genes are not expressed ordinarily.

Figure 3

Figure 3

. De novo synthesis of ATP and GTP. The enzymes encoded by the different genes are guaB, IMP dehydrogenase; guaA, GMP synthase; gmk, GMP kinase; ndk, nucleoside diphosphokinase; purA, adenylosuccinate synthetase; purB, adenylosuccinate lyase; and adk (more...)

Adenine and guanine nucleotides can be inter-converted through the common precursor IMP. These conversions serve to balance both types of nucleotides in the cellular pool and play an important role when the bacterium can salvage nucleobases in its habitat (40). Adenine nucleotides can be converted to guanine nucleotides via two pathways for which orthologous genes have not been found in H. pylori DNA. GMP is converted to IMP by the action of GMP reductase, which seems to be encoded in H. pylori DNA by the gene HP0854 orthologous of guaC.

The results of studies of de novo purine biosynthesis in H. pylori suggest that this pathway is essential to the bacterium and emphasize the need to compare the conclusions of genomic analysis with metabolic data in the quest to understand the physiology of the bacterium.

Salvage of Preformed Purines

Many organisms that are able to synthesize purines de novo also are able to scavenge a wide range of purines, convert them to other purines, and incorporate them into nucleic acids (37). Salvage routes for purine nucleotide synthesis in H. pylori were investigated by measuring the incorporation of adenine and guanine and the activities of salvage enzymes (Fig. 4). Radiolabeled adenine and guanine were incorporated at rates of 64.5 and 27.5 pmol h−1 (106 cells)−1 (18). Phosphoribosyl transferases are key enzymes in the salvage of purines, and significant adenine-, guanine-, and hypoxanthine phosphoribosyl transferase activities are observed in H. pylori lysates (18). The genes HP0572 orthologous to apt encoding adenine phosphoribosyl transferase in E. coli and HP0735 orthologous to gpt coding for a xanthine/guanine phosphoribosyl transferase (XGPRTase) in H. influenzae were identified in H. pylori DNA. The rate measured for H. pylori HPRTase activity converting hypoxanthine to IMP was significantly lower than those for APRTase and GPRTase (18), and no orthologous gene for a specific HPRTase was found in the H. pylori genome. In E. coli and S. enterica serovar Typhimurium, the genes gpt and hpt encode GPRTase and HPRTase, respectively. However, in H. influenzae, hypoxanthine, xanthine, and guanine are substrates for XGPRTase to synthesize IMP, XMP, and GMP, respectively; and in B. subtilis, the same transferase uses guanine or hypoxanthine (23). Thus, it is possible that the enzyme encoded by the gpt gene in H. pylori is able to utilize both guanine and hypoxanthine. Interestingly, there is a relationship between the uptake of adenine, guanine, and hypoxanthine and the activities of the corresponding phosphoribosyltransferases in H. pylori, suggesting that uptake mechanisms are under the same metabolic controls as the salvage biosynthesis pathways, similar to what occurs in E. coli, S. enterica serovar Typhimurium, and B. subtilis. The significant amounts of purine bases incorporated by H. pylori and the relatively high activities measured for the transferases indicate that the bacterium can salvage purines efficiently via this pathway.

Figure 4

Figure 4

. Salvage and interconversion of purines. The enzymes encoded by the different genes are deoD, purine nucleoside phosphorylase; apt, adenine phosphoribosyl transferase; purB, adenylosuccinate lyase; purA, adenylosuccinate synthetase; guaC, GMP reductase; (more...)

A route for the synthesis of IMP or GMP from inosine or guanosine, respectively, involves the phosphorylation of the corresponding nucleoside. In E. coli and S. enterica serovar Typhimurium, the enzyme catalyzing this step is guanosine kinase encoded by gsk. The absence of guanosine kinase activity (18), and of a gene coding for it (3, 33), indicates that this route is not present in H. pylori. In E. coli and S. enterica serovar Typhimurium, a major pathway for the salvage of purine nucleosides is their degradation to the corresponding base by a purine-nucleoside phosphorylase encoded by deoD, followed by the conversion to a nucleotide monophosphate by the appropriate phosphoribosyltransferases (40) (Fig. 4). No purine-nucleoside phosphorylase activity was detected in H. pylori (18), although gene HP1178 orthologous to deoD was identified in its genome (3, 33). This discrepancy may be explained by the relatively high adenine and guanine nucleosidase activities observed in H. pylori cell extracts, which hydrolyze the nucleosides to ribose and the corresponding base and could substitute for purine-nucleoside phosphorylase in the hydrolysis of the nucleosides. Nucleosidase activities may have masked phosphorylase activities, since the method employed to assess the presence of the latter enzyme measures its activity at the same time as those of the nucleosidases (18). Alternatively, deoD may not be expressed under the bacterial growth conditions employed in that study. In E. coli and S. enterica serovar Typhimurium, the synthesis of purine-nucleoside phosphorylase is induced by purine nucleosides in the growth medium with concomitant suppression of the contributions of the de novo pathway to the purine nucleotide pool (40).

It is of interest that considerable purine nucleoside phosphotransferase activities were measured in H. pylori cell extracts (18). These enzymes phosphorylate adenosine or guanosine to AMP or GMP, respectively, and may constitute an alternative nucleoside salvage pathway not found in E. coli, S. enterica serovar Typhimurium, or B. subtilis. No genes encoding these enzymes have been identified in the H. pylori genome (3, 33).

The significant activities of adenosine nucleosidase and adenine phosphoribosyltransferases, the lower activity of adenosine phosphotransferase, and the lack of adenosine kinase activity suggest that the principal route for adenosine utilization in H. pylori is via the salvage of the purine ring after hydrolysis of adenosine to adenine, and phosphorylation by adenine phosphoribosyltransferases, although some production of AMP would also occur by direct phosphorylation by adenosine phosphotransferase. Similar mechanisms operate for the utilization of guanosine (18). Thus, the catabolism of ribonucleosides and deoxyribonucleosides in H. pylori appears to follow similar pathways as in E. coli and S. enterica serovar Typhimurium. In these bacteria the nucleosides are not phosphorylated directly but are first degraded to free bases that react with PRPP to yield the corresponding nucleoside monophosphate derivative (40).

The high activities observed for adenylate kinase and its ability to phosphorylate AMP using ATP, GTP, or ITP suggest the presence in H. pylori of an effective way of contributing to the balance of intracellular purine nucleotide pools. The absence of detectable hydrolysis of AMP by H. pylori lysates indicates very low activities of AMP hydrolase and AMP nucleosidase, if at all present. The synthesis of coenzymes containing the adenylate moiety is an important metabolic role of purine nucleotides; thus, transfer of phosphate groups from GTP or ITP to AMP in H. pylori may be a mechanism used by the bacterium to keep the necessary supply of flavin nucleotides, nicotinamide nucleotides, and coenzyme A.

Biosynthesis of Deoxyribonucleotides

The ratio of RNA to DNA in cells is between 5:1 and 10:1, indicating that most of the carbon that flows through the nucleotide biosynthesis pathways is directed to the ribonucleoside triphosphate (rNTP) pools. Nonetheless, the relatively small fraction that is directed to synthesizing deoxyribonucleoside triphosphates (dNTPs) is of fundamental importance for the life of the cell since dNTPs are employed almost exclusively in the biosynthesis of DNA. In most organisms, the first step specific for deoxyribonucleotide synthesis is catalyzed by ribonucleoside diphosphate reductase (rNDPase), which converts all four ribonucleoside diphosphates to the corresponding 2′-deoxyribonucleoside diphosphates. In H. pylori, the genes HP0680 and HP0364 have similarities with the nrdA gene from E. coli encoding the α subunit of this enzyme and the nrdB gene from Synechocystis sp., which encodes the β subunit, respectively.

The biosynthesis of thymidine triphosphate (dTTP) occurs partly from the dUDP produced by rNDPase and partly from deoxycytidine nucleotides. In E. coli, dTTP synthesis requires four additional steps catalyzed by dUTPase (dUTP→dUMP) encoded by dut, thymidylate synthase (dUMP → dTMP) encoded by thyA, dTMP kinase (dTMP → dTDP) encoded by tmk, and nucleoside diphosphokinase (dTDP→dTTP) encoded by ndk (Fig. 5). In H. pylori DNA, the genes HP0865, HP1474, and HP0198 are orthologous to dut, tmk, and ndk, respectively; but no gene orthologous to thyA has been identified (3, 33).

Figure 5

Figure 5

. Biosynthesis of deoxyribonucleotides. The enzymes encoded by the different genes are nrd, ribonucleoside diphosphate reductase; ndk, nucleoside diphosphokinase; dcd, dCTP deaminase; dut, deoxyuridine triphosphatase; thyA, thymidylate synthase; tmk, (more...)

The dTTP salvage pathway described in E. coli requires the presence of uridine phosphorylase or thymidine phosphorylase, and thymidine kinase, encoded by udp, deoA, and tdk genes, respectively (21). No genes sharing similarity with these genes were found in H. pylori (3, 33). Alternatively, dTTP may be provided by the action of exodeoxyribonuclease on DNA releasing nucleotide 5′-monophosphates, which can be converted to triphosphates as described above. The gene HP1526 is similar to the lexA gene encoding this enzyme in B. subtilis.

The salvage route for pyrimidine deoxynucleotide synthesis in H. pylori was investigated by measuring the incorporation of deoxycytidine and thymidine and the activities of the corresponding kinases (17). It is of interest to note that although the bacterium does not appear to take up either deoxynucleoside in any significant amounts, and the genes encoding thymidine phosphorylase (deoA) and thymidine kinase (tdk) have not been identified in the genome (3, 33), the latter enzyme activity is detected in lysates (17). The role of this activity in H. pylori is unclear, and it is possible that the observed thymidine kinase activity reflects a relaxed specificity of a deoxypurine nucleoside kinase.

H. pylori salvages deoxyadenosine but at much lower rates than adenosine, and any incorporation of deoxyguanosine is below the detection levels of the radioactive tracer experimental techniques employed (18). Nucleosidases hydrolyze deoxynucleosides to deoxyribose and the corresponding base, and nucleoside phosphotransferases produce deoxynucleoside monophosphates by phosphorylation of deoxynucleosides. Significant deoxyadenosine nucleosidase and phosphotransferase activities, as well as deoxyguanosine phosphotransferase activity, but no deoxyguanosine nucleosidase activity are measured in H. pylori (18). The incorporation of deoxyadenosine by H. pylori, together with the presence of deoxyadenosine phosphotransferase, suggests that phosphorylation of the deoxynucleoside is a pathway for the synthesis of adenosine deoxynucleotides (18). However, the operation of this salvage mechanism is limited by the presence of an equally active deoxyadenosine nucleosidase, which would partly reroute the purine base moiety via some of the other biosynthetic pathways. The lack of incorporation of deoxyguanosine and the absence of deoxyguanosine nucleosidase activity suggest that the synthesis of guanosine deoxynucleotides follows different pathways, notwithstanding the deoxyguanosine phosphotransferase activity observed. It may be possible that some of the deoxyribose produced by the action of deoxyadenosine nucleosidase is recycled into the synthesis of guanine deoxyribonucleotides (18).


There are only a few studies on the nucleotide metabolism of H. pylori, but together with the information derived from analyses of the genome of the bacterium, they have provided a wealth of information about the pathways of biosynthesis and degradation of pyrimidine and purine nucleotides, and showed that nucleotide biosynthetic enzymes are potential targets for antimicrobials designed against this organism.

H. pylori synthesizes pyrimidine nucleotides de novo and this pathway is essential for the growth and survival of the bacterium. This finding is in agreement with its limited capacity to salvage pyrimidines and the absence from its genome of key enzymes of pyrimidine salvage pathways. H. pylori can survive and proliferate by synthesizing de novo purine nucleotides, but it has yet to be established whether this pathway is essential for the bacterium. The organism incorporates preformed purines, and pathways for purine salvage are clearly identifiable in its genome. Although these salvage pathways act as energy-saving devices by utilizing the preformed purine ring and the ribose moiety of purine nucleosides, it has yet to be elucidated whether H. pylori can survive by having recourse only to purine salvage; and there is some evidence that these pathways may not be sufficient to support the integrity and proliferation of the bacterium.

Studies have demonstrated that targeting the inhibition of the de novo pyrimidine pathway presents valuable opportunities for the development of antimicrobial agents selective for H. pylori. The potential of the pathways of purine synthesis as therapeutic targets for antibacterials specific to H. pylori has yet to be explored, and to achieve this it is necessary to obtain a better understanding of the metabolism of purine nucleotides in the bacterium.


Adair L. B., Jones M. E. Purification and characteristics of aspartate transcarbamylase from Pseudomonas fluorescens. J. Biol. Chem. 1972;247:2308–2315. [PubMed: 4336369]
Ahonkhai I., Kamekura M., Kushner D. J. Effects of salts on the aspartate transcarbamylase of a halophilic bacterium, Vibrio costicola. Biochem. Cell Biol. 1989;67:666–669.
Alm R. A., Ling L. S., Moir D. T., King B. L., Brown E. D., Doig P. C., Smith D. R., Noonan B., Guild B. C., deJonge B. L., Carmel G., Tummino P. J., Caruso A., Uria-Nickelsen M., Mills D. M., Ives C., Gibson R., Merberg D., Mills S. D., Jiang Q., Taylor D. E., Vovis G. F., Trust T. J. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature. 1999;397:176–180. [PubMed: 9923682]
Bergh S. T., Evans D. R. Subunit structure of class A aspartate transcarbamylase from Pseudomonas fluorescens. Proc. Natl. Acad. Sci. USA. 1993;90:9818–9822. [PMC free article: PMC47663] [PubMed: 8234318]
Bethell M. R., Jones M. E. Molecular size and feedback-regulation of bacterial aspartate transcarbamylase. Arch. Biochem. Biophys. 1969;134:352–365. [PubMed: 4311178]
Burns B. P., Hazell S. L., Mendz G. L. In situ properties of aspartate carbamoyltransferase activity in Helicobacter pylori. Arch. Biochem. Biophys. 1997;347:119–125. [PubMed: 9344472]
Burns B. P., Hazell S. L., Mendz G. L. A novel mechanism for resistance to the antimetabolite N-phosphonoacetyl-l-aspartate by Helicobacter pylori. J. Bacteriol. 1998;180:5574–5579. [PMC free article: PMC107614] [PubMed: 9791105]
Burns B. P., Hazell S. L., Mendz G. L., Kolesnikow T., Tillett D., Neilan B. A. The Helicobacter pylori pyrB gene encoding aspartate carbamoyltransferase is essential for survival. Arch. Biochem. Biophys. 2000;380:78–84. [PubMed: 10900135]
Chang T.-Y., Jones M. E. Aspartate transcarbamylase from Streptococcus faecalis. Purification, properties, and nature of an allosteric activator site. Biochemistry. 1974;13:629–638. [PubMed: 4204271]
Chu C., West T. P. Pyrimidine biosynthetic pathway of Pseudomonas fluorescens. J. Gen. Microbiol. 1990;136:875–880. [PubMed: 1974280]
Copeland R. A., Marcinkeviciene J., Haque T. S., Kopcho L. M., Jiang W., Wang K., Ecret L. D., Sizemore C., Amsler K. A., Foster L., Tadesse S., Combs A. P., Stern A. M., Trainor G. L., Slee A., Rogers M. J., Hobbs F. Helicobacter pylori-selective antibacterials based on inhibition of pyrimidine biosynthesis. J. Biol. Chem. 2000;275:33373–33378. [PubMed: 10938275]
Grem J. L., King S. A., O'Dwyer P. J., Leyland-Jones B. Biochemistry and clinical activity of N-(phosphonacetyl)-l-aspartate: a review. Cancer Res. 1988;48:4441–4454. [PubMed: 3293772]
Guy H. I., Evans D. R. Cloning and expression of the mammalian multifunctional protein CAD in Escherichia coli. Characterization of the recombinant protein and a deletion mutant lacking the major interdomain linker. J. Biol. Chem. 1994;269:23808–23816. [PubMed: 7916346]
Jyssum S., Jyssum K. Metabolism of pyrimidine bases and nucleosides in Neisseria meningiditis. J. Bacteriol. 1979;138:320–323. [PMC free article: PMC218180] [PubMed: 108255]
Kaneko T., Tanako A., Sato S., Kotani H., Sazuka T., Miyajima N., Sugiura M., Tabata S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. I. Sequence features in 1 Mb region from map position 64% to 92% of the genome. DNA Res. 1997;2:153–166. [PubMed: 8590279]
Kenny M. J., McPhail D., Shepherdson M. A reappraisal of the diversity and class distribution of aspartate trans-carbamylases in gram-negative bacteria. Microbiology. 1996;142:1873–1879. [PubMed: 8757751]
Mendz G. L., Jimenez B. M., Hazell S. L., Gero A. M., O'Sullivan W. J. De novo synthesis of pyrimidine nucleotides by Helicobacter pylori. J. Appl. Bacteriol. 1994;77:1–8. [PubMed: 7928775]
Mendz G. L., Jimenez B. M., Hazell S. L., Gero A. M., O'Sullivan W. J. Salvage synthesis of purine nucleotides by Helicobacter pylori. J. Appl. Bacteriol. 1994;77:674–681. [PubMed: 7822226]
Mendz G. L., Shepley A. J., Hazell S. L. Survival of Helicobacter pylori by de novo synthesis of pyrimidine nucleotides. Gut Suppl. 1996;39:A73.
Mendz G. L., Shepley A. J., Hazell S. L., Smith M. A. Purine metabolism and the microaeropohily of Helicobacter pylori. Arch. Microbiol. 1997;168:448–456. [PubMed: 9385135]
Neuhard, J., and R. A. Kelln. 1996. Biosynthesis and conversion of pyrimidines, p. 580–599. In F. C. Neidhart (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. American Society for Microbiology, Washington, D.C.
Neumann J., Jones M. E. End-product inhibition of aspartate transcarbamylase in various species. Arch. Biochem. Biophys. 1964;104:438–447. [PubMed: 14161013]
Nygaard, P. 1993. Purine and pyrimide salvage pathways, p. 359–378. In A. L. Sonenshein (ed.), Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics. American Society for Microbiology, Washington, D.C.
O'Donovan G. A., Neuhard J. Pyrimidine metabolism in microorganisms. Bacteriol. Rev. (1970);34:278–343. [PMC free article: PMC378357] [PubMed: 4919542]
Pragobpol S., Gero A. M., Lee C. S., O'Sullivan W. J. Orotate phosphorybosyltransferase and orotidylate decarboxylase from Crithidia luciliae. Subcellular location of the enzymes and evidence for substrate channeling. Arch. Biochem. Biophys. 1984;230:285–293. [PubMed: 6712237]
Purcarea C., Erauso G., Prieur D., Hervé G. Aspartate transcarbamylase from the deep-sea hyperthermophile archeon Pyrococcus abyssi: genetic organisation, structure, and expression in Escherichia coli. Microbiology. 1994;140:1967–1975.
Quinn C. L., Stephenson B. T., Switzer R. L. Functional organisation and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. J. Biol. Chem. 1991;266:9113–9127. [PubMed: 1709162]
Reynolds D. J., Penn C. W. Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology. 1994;140:2649–2656. [PubMed: 8000535]
Roland K. L., Powell F. E., Turnbough C. L. Role of translation and attenuation in the control of pyrBI expression in Escherichia coli. J. Bacteriol. 1985;163:991–999. [PMC free article: PMC219230] [PubMed: 3928602]
Roth, B. 1983. Selective inhibitors of bacterial dihydrofolate reductase: structure-activity relationships, p. 107–127. In G. H. Hitchings (ed.), Inhibition of Folate Metabolism in Chemotherapy. Springer-Verlag, Berlin, Germany.
Shepley, A. J., G. L. Mendz, and S. L. Hazell. 1995. The essential role of de novo pyrimidine nucleotide synthesis in Helicobacter pylori, abstr. P-58. In Proc. 7th FAOBMB Congress. Australian Society for Biochemistry and Molecular Biology, Sydney, Australia.
Swyryd E. A., Seaver S. S., Stark G. R. N-(phosphonacetyl)-l-aspartate, a potent transition state analog inhibitor of aspartate transcarbamylase, blocks proliferation of mammalian cells in culture. J. Biol. Chem. 1974;249:6945–6950. [PubMed: 4371054]
Tomb J. F., White O., Kerlavage A. R., Clayton R. A., Sutton G. G., Fleishmann R. D., Ketchum K. A., Kienk H. P., Gill S., Dougherty B. A., Nelson K., Quackenbuch J., Zhou L., Kirkness E. F., Peterson S., Loftus B., Richardson D., Dodson R., Khalk H. G., Glodek A., McKenney K., Fitzgerald L. M., Lee M., Adams M. D., Hickey E. K., Berg D. E., Gocayne I. D., Fujii C., Bowman C., Watthey L., Wallin E., Hayes W. S., Borodovsky M., Karp P. D., Smith H. O., Fraser C. M., Venter J. C. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 1997;388:539–547. [PubMed: 9252185]
Traut T. W., Jones M. E. Inhibitors of orotate phosphoribosyl-transferase and orotidine-5′-decarboxylase from mouse Ehrlich ascites cells: a procedure for analyzing the inhibition of a multi-enzyme complex. Biochem. Pharmacol. 1977;26:2291–2296. [PubMed: 563234]
Turner R. J., Lu Y., Switzer R. L. Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism. J. Bacteriol. 1994;176:3708–3722. [PMC free article: PMC205560] [PubMed: 8206849]
Umezu K., Amaya T., Yoshimoto A., Tomita K. Purification and properties of orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase from baker's yeast. J. Biochem. (Tokyo) 1971;70:249–262. [PubMed: 5094209]
Wheeler P. R. Recent research into the physiology of Mycobacterium leprae. Adv. Microb. Physiol. 1990;31:71–124. [PubMed: 2264525]
Wild J. R., Belser W. L. Pyrimidine biosynthesis in Serratia marcescens: a possible role for nonsequential enzyme interactions in mimicking coordinate gene expression. Biochem. Genet. 1977;15:157–172. [PubMed: 192191]
Wild J. R., Belser W. L. Pyrimidine biosynthesis in Serratia marcescens: polypeptide interactions of three nonsequential enzymes. Biochem. Genet. 1977;15:173–180. [PubMed: 322653]
Zalkin, H., and P. Nygaard. 1996. Biosynthesis of purine nucleotides, p. 561–579. In F. C. Neidhart (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. American Society for Microbiology, Washington, D.C.
Copyright © 2001, ASM Press.
Bookshelf ID: NBK2414PMID: 21290716
PubReader format: click here to try


  • PubReader
  • Print View
  • Cite this Page

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to pubmed
  • Taxonomy
    Related Taxonomy Entries
  • Gene
    Gene records cited in chapters on the NCBI bookshelf. Links are provided by the authors or the NCBI Bookshelf staff.

Related citations in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...