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

. 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...)

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.

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

. 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

. 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

. 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.