Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Presence of Nonoxidative Enzymes of the Pentose Phosphate Shunt in Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-¹⁴C]ribose into CO₂ and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.     

Ribose biosynthesis and evidence for an alternative first step in the common aromatic amino acid pathway in Methanococcus maripaludis.

An acetate-requiring mutant of Methanococcus maripaludis allowed efficient labeling of riboses following growth in minimal medium supplemented with [2-(13)C]acetate. Nuclear magnetic resonance and mass spectroscopic analysis of purified cytidine and uridine demonstrated that the C-1' of the ribose was about 67% enriched for 13C. This value was inconsistent with the formation of erythrose 4-phosphate (E4P) exclusively by the carboxylation of a triose. Instead, these results suggest that either (i) E4P is formed by both the nonoxidative pentose phosphate and triose carboxylation pathways or (ii) E4P is formed exclusively by the nonoxidative pentose phosphate pathway and is not a precursor of aromatic amino acids.     

Defective formation and/or utilization of carbon monoxide in H2/CO2 fermenting methanogens dependent on acetate as carbon source

Autotrophic methanogens reduce CO₂ to CO and assimilate CO in a carbonylation reaction. Heterotrophic species were found not to form CO and/or to incorporate CO into cell matiral. The absence of CO formation correlated with the absence of carbon monoxide dehydrogenase activity. The heterotrophic Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanococcus voltae and Methanospirillum hungatei (strain GP 1) were investigated.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Enzymatic Studies on the Conversion of Erythritol Fermentation to d-Mannitol Fermentation in n-Alkane-grown Yeast Candida zeylanoides

In the polyol fermentation by Candida zeylanoides KY6166, which occurred preferentially by keeping the pH of medium at acidic side (below 4.0), phosphate ion played a precise role in the conversion of erythritol fermentation to d-mannitol fermentation. Enzymatic studies on the conversion mechanism provided the following evidences.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphate ion.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphateion. From these results, the alteration from erythritol fermentation to mannitol fermentation by phosphate ion was explained as the result of the change in the level of erythrose 4-phosphate and fructose 6-phosphate which was caused by the inhibition of transketolase.     

Pyruvate oxidation by Methanococcus spp.

In the absence of H₂, Methanococcus spp. utilized pyruvate as an electron donor for methanogenesis. For Methanococcus voltae A3, Methanococcus maripaludis JJ1, and Methanococcus vannielii, typical rates of pyruvate-dependent methanogenesis were 3.4, 2.8, and 3.9 nmol min^-1 mg^-1 cell dry wt, respectively. These rates were 1–4% of the rates of H₂-dependent methanogenesis. For M. voltae, the concentration of pyruvate required for one-half the maximum rate of methanogenesis was 7 mM, and pyruvate-dependent methanogenesis was linear for 3 days. Radiolabeled acetate was formed from [3-¹⁴C]pyruvate, and the stoichiometry of pyruvate consumed per acetate produced was 1.12±0.27. The stoichiometry of pyruvate consumed per CH₄ produced was 3.64±0.34. These values are close to the expected values of 1 acetate and 4 CH₄. Although 10–30% of total cell carbon could be obtained from exogenous pyruvate during growth with H₂, pyruvate did not replace the nutritional requirement for acetate in Methanococcus voltae A3 or two acetate auxotrophs of Methanococcus maripaludis, JJ6 and JJ7. These results suggest that pyruvate was not oxidized in the presence of H₂. The inability to oxidize pyruvate during H₂-dependent methanogenesis would prevent a futile cycle of pyruvate oxidation and biosynthesis during autotrophic growth.     

Subcellular distribution of enzymes of the oxidative pentose phosphate pathway in root and leaf tissues

The subcellular distribution of enzymes of the oxidative pentose phosphate pathway was studied in plants. Root and leaf tissues from several species were separated by differential centrifugation into plastidic and cytosolic fractions. In all tissues studied, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in both plastidic and cytosolic compartments. In maize and pea root, and spinach and pea leaf, the non-oxidative enzymes of the pentose phosphate pathway (transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to the plastid. In tobacco leaf and root, however, the non-oxidative enzymes were found in the cytosolic as well as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was supplied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, indicating the presence of a translocator capable of transporting pentose phosphates.Keywords: Pentose phosphate, subcellular, plastid, ribulose 5-phosphate, compartmentation      

Cell-free synthesis and membrane-integration of the reaction center subunit H from Rhodobacter capsulatus

The flagellins of Methanospirillum hungatei strains JF1 and GP1, Methanococcus deltae, and Methanothermus fervidus are glycosylated. Isolated flagellar filaments from these organisms are dissociated by low concentrations (0.5% (v/v)) of Triton X-100. Flagellar filaments from other methanogens (Methanococcus voltae, Methanococcus vannielii and Methanoculleus marisnigri) composed of non-glycosylated flagellins are resistant to Triton X-100 treatment. Consequently, the isolation techniques (employing Triton X-100) used to isolate basal body-hook-filament complexes in eubacteria may not be applicable to many methanogens.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等.葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中.本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用.     

Biosynthetic pathways in Methanospirillum hungatei as determined by 13C nuclear magnetic resonance

The main metabolic pathways in Methanospirillum hungatei GP1 were followed by using 13C nuclear magnetic resonance, with 13C-labeled acetate and CO2 as carbon sources. The labeling patterns found in carbohydrates, amino acids, lipids, and nucleosides were consistent with the formation of pyruvate from acetate and CO2 as the first step in biosynthesis. Carbohydrates are formed by the glucogenic pathway, and no scrambling of label was observed, indicating that the oxidative or reductive pentose phosphate pathways are not functioning at significant rates. The pathways for amino acid biosynthesis are the usual ones, with the exception of that for isoleucine. The tricarboxylic acid pathway is incomplete and operates in a reductive direction to form alpha-ketoglutarate. The phytanyl chains of lipids are synthesized from acetate via mevalonic acid.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等。葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中。本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用。     

Presence of nonoxidative enzymes of the pentose phosphate shunt in Tetrahymena.

Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-14C]ribose into CO2 and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.     

The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle

Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, α-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known ;overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30-40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine-zinc-insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine-zinc-insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding.     

Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria

Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-¹³C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-¹³C]-, [2-¹³C]-, or [3-¹³C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.     

Relatedness of the flagellins from methanogens

Purified flagellar filaments isolated from six methanogens were composed of multiple flagellins. Two flagellins were present in Methanococcus deltae (M _r =34000 and 32000), Methanoculleus marisnigri (M _r =31000 and 25500) and Methanococcus jannaschii (M _r =31000 and 27500), three in Methanothermus fervidus (M _r =34000, 25000 and 24000) and four or more in both Methanococcus vanniellii and Methanococcus maripaludis (M _r ranging from 27500 to 32000). The flagellins of M. fervidus and M. deltae reacted positively with glycoprotein-specific stains. The flagellins of M. deltae, M. maripaludis and M. vannielii were closely related to those of M. voltae based on cross-reactivity with antisera raised against M. voltae flagellins and homology with flagellin-specific oligonucleotide probes to the N-terminus and leader peptide of M. voltae flagellins. Similarities appear to exist among the flagellins of M. fervidus, M. marisnigri and Halobacterium halobium based on cross-reactivity with antisera produced against the flagella of Methanospirillum hungatei JF1. The N-termini of the flagellins from the mesophilic Methanococcus spp. and M. marisnigri show homology with the N-termini of other archaebacterial flagellins. These N-termini may undergo a modification involving removal of a leader peptide.     

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an <Emphasis Type="Italic">E. coli</Emphasis> strain disabled in the oxidative pentose phosphate pathway

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (¹³C, ¹⁵N, and/or ²H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-¹³C-glycerol and ¹⁵N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, ¹³C-sodium formate combined with ¹³C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to ¹J_C5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of ¹³C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and ¹⁵N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.     

Levels of Water-Soluble Vitamins in Methanogenic and Non-Methanogenic Bacteria

The levels of seven water-soluble vitamins in Methanobacterium thermoautotrophicum, Methanococcus voltae, Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Bacteroides thetaiotaomicron were compared by using a vitamin-requiring Leuconostoc strain. Both methanogens contained levels of folic acid and pantothenic acid which were approximately two orders of magnitude lower than levels in the nonmethanogens. Methanobacterium thermoautotrophicum contained levels of thiamine, biotin, nicotinic acid, and pyridoxine which were approximately one order of magnitude lower than levels in the nonmethanogens. The thiamine level in Methanococcus voltae was approximately one order of magnitude lower than levels in the nonmethanogens. Only the levels of riboflavin (and nicotinic acid and pyridoxine in Methanococcus voltae) were approximately equal in the methanogens and nonmethanogens. Folic acid may have been present in extracts of methanogens merely as a precursor, by-product, or hydrolysis product of methanopterin.     

Biosynthesis of 5-hydroxybenzimidazolylcobamid (factor III) in Methanobacterium thermoautotrophicum.

Cultures of Methanobacterium thermoautotrophicum were supplemented with 13C-labeled acetate or pyruvate, and the labeling pattern of the corrinoid, factor III, was established by 13C NMR spectroscopy. Complete 13C signal assignments were obtained by two-dimensional NMR experiments. The labeling pattern of factor III was analyzed by comparison with those of amino acids and nucleosides. The corrin ring system is derived from eight molecules of glutamate. The aminopropanol moiety is derived in a hitherto unknown pathway from pyruvate by reductive amination. The heterocyclic ring of hydroxybenzimidazole shares the labeling pattern of the imidazole ring of purines. The remaining four carbon atoms of the carbocyclic ring show the labeling signature of a carbohydrate with two of the carbons introduced from acetate and two from C-1 of pyruvate. However, erythrose can be ruled out as the specific precursor on the basis of a detailed investigation of aromatic amino acids indicating that erythrose 4-phosphate is obtained by reductive carboxylation of a triose precursor and not by the pentose phosphate cycle.