Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Regulation of nitrogenase activity by covalent modification in Chromatium vinosum

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH ₄ ⁺ . Activity of the Fe protin component of nitrogenase required both Mn²⁺ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.Abbreviation HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid     

Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C₄-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO₂ in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

The Relationship of Cobalt Requirement to Vitamin B12-dependent Methionine Synthesis in Rhizobium meliloti

As a growth factor, Rhizobium meliloti required cobalt ion, or vitamin B₁₂ which was found to be incorporated into the cells without decomposition to cobalt ion. Trial of replacement for cobalt ion by the addition of various compounds to the cobalt-deficient medium revealed that methionine could substitute for cobalt ion and promote the growth in response to its concentration. Furthermore, B₁₂-dependent methionine synthetase was demonstrated in the cell-free extracts of this microorganism. The morphological change of R. meliloti by the additions to the medium was observed microscopically.     

Characterization of a Vitamin B12 Compound in the Edible Purple Laver,Porphyra yezoensis

The edible purple laver, Porphyra yezoensis, contained 51.49±1.51 μg of vitamin B₁₂ compounds per 100 g dry weight of the laver (mean±SEM, n=4). A vitamin B₁₂ compound was purified from the lyophilized purple laver and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B₁₂, but not to those of vitamin B₁₂ analogues inactive for humans.     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope fractionation technique

1. The carbon isotope discrimination properties of a representative of each of the three types of photosynthetic bacteria Chlorobium thiosulfatophilum, Rhodospirillum rubrum and Chromatium and of the C₃-alga Chlamydomonas reinhardii were determined by measuring the ratio of ¹³CO₂ to ¹²CO₂ incorporated during photoautotrophic growth. 2. Chromatium and R. rubrum had isotope selection properties similar to those of C₃-plants, whereas Chlorobium was significantly different. 3. The results suggest that Chromatium and R. rubrum assimilate CO₂ mainly via ribulose 1,5-diphosphate carboxylase and the associated reactions of the reductive pentose phosphate cycle, whereas Chlorobium utilizes other mechanisms. Such mechanisms would include the ferredoxin-linked carboxylation enzymes and associated reactions of the reductive carboxylic acid cycle.Abbreviations RuDP ribulose 1,5-disphosphate - PEP phosphoenolpyruvate     

Purification and Characterization of S-adenosylmethionine Synthetase from Wheat Embryos: Subunit Structure and Molecular Properties

S-adenosylmethionine synthetase from wheat embryos was purified to electrophoretic homogeneity. The mol wt of the enzyme was 174,000 as determined by molecular sieve chromatography on Sephacryl S-200. A single subunit of purified AdoMet synthetase was observed on SOS-PAGE with a mol wt of 84,000 suggesting that the enzyme is a homodimer. The apparent Km of purified enzyme with ATP and methionine is 80 μM and 100 μM, respectively. The pH optimum of the enzyme is 7.75. The enzyme requires MgCb, KCI and reduced glutathione for optimum activity. The ³H-labelled putative S-adenosylmethionine reaction product was converted into ³H-labelled 5′-methyl-thioadenosine by heat treatment (100°C, 10 min, pH 7.0). This proved the authenticity of the reaction product of the AdoMet synthetase in wheat embryos.     

Characterization of a Corrinoid Compound in the Edible (Blue-Green) Alga,Suizenji-nori

The edible blue-green alga (cyanobacterium), Suizenji-nori, contained 143.8±22.4 μg of vitamin B₁₂ per 100 g dry weight of the alga (mean±SE, n=4). A corrinoid compound was purified from the dried Suizenji-nori, and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified corrinoid compound were not identical to those of true vitamin B₁₂, but to those of pseudovitamin B₁₂ which is inactive for humans.     

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

Cobalamin (vitamin B₁₂) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B₁₂ biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG^AcbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B₁₂ riboswitch upstream of the cbiXJCDETLFGAcysG^AcbiYbtuR operon and the recombinant production of three different vitamin B₁₂ binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B₁₂-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B₁₂ concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.     

Oxidation of compounds metabolized through folate coenzyme pathways in vitamin B12-deficient rats

The effects of vitamin B₁₂ deficiency in rats and dietary supplementation with vitamin B₁₂ and/or l-methionine plus folate on the oxidation of compounds metabolized through folate coenzyme pathways were investigated. Rats fed a vitamin B₁₂-deficient diet oxidized significantly lower amounts in 60 min of l-histidine, glycine, sarcosine, formate, and l-serine to CO₂ than vitamin B₁₂-supplemented controls. Supplementation of the deficient diet with l-methionine plus folate restored the ability to oxidize the ring-2-carbon of l-histidine, the methyl group of sarcosine, and formate to the same level as that observed in animals receiving vitamin B₁₂. In contrast, oxidation of the 1-carbon of glycine and the 3-carbon of l-serine was not restored to control levels by addition of methionine plus folate to the vitamin B₁₂-deficient diet. Inhibition of the metabolism of the 2-carbon of glycine to CO₂ was partially overcome by additional dietary methionine and folate. Glycine synthase activity in homogenates paralleled the in vivo pattern of oxidation of the 1-carbon of glycine to CO₂, whereas sarcosine dehydrogenase activity appeared to increase 2-fold in vitamin B₁₂ deficiency.     

Dependence of Betaine Stimulation of Vitamin B12 Overproduction on Protein Synthesis

The betaine-stimulated differential synthesis of vitamin B₁₂, i.e., the increase in B₁₂ per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, δ-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B₁₂ synthesis is a result of its stimulation of synthesis of δ-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B₁₂ and δ-aminolevulinic acid synthetase formation, no matter when it was added to the system.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

ENZYMES CATALYZING THE DE NOVO SYNTHESIS OF METHYL GROUPS IN THE BRAIN AND OTHER TISSUES OF THE RAT

—Rat brain contains all three of the enzymes required for de novo synthesis of the methyl group of methionine (serine transhydroxymethylase, methylene reductase, and [B₁₂]transmethylase) in activities comparable to those found in liver and kidney. The activities of methylene reductase in female kidney, and of [B12]transmethylase in female brain and kidney, are higher than in the corresponding male tissues. Liver and kidney extracts contain an inhibitor of methylene reductase not present in brain extracts. This inhibitor differs from S-adenosylmethionine (SAM), which also inhibits methylene reductase in both liver and brain homogenates. The administration of l -DOPA to rats, which has been previously shown to deplete brain S-adenosylmethionine, also reduces the activity of brain [B₁₂]transmethylase if assayed without added SAM. Since SAM is required for activity of this enzyme, its decreased activity probably results from the decline in brain SAM concentration. De now synthesis of methyl groups could be a mechanism by which the brain maintains its level of methionine in the face of increased methyl group utilization after administration of l -DOPA.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine.

The effects of media vitamin B₁₂(CNB₁₂), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH₄folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mm methionine elevates the amount of B₁₂ methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μm. Conversely, excess 100 μm folic acid increases the amount of B₁₂ holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 mm methionine. These increases in B₁₂ methyltransferase promoted by 100 μm media folate and 10 mm methionine are inhibited by cycloheximide. 5-MeH₄folate will support growth and induce methyltransferase synthesis more efficiently than folic acid.Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 μm CNB₁₂, 0.1 mm homocysteine, and 100 μm folic acid or 10 μm dl-5-MeH₄folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB₁₂ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mm methionine-containing to methionine-free media is dependent upon the B₁₂ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μm CNB₁₂ to stabilize and convert apo- to holomethyltransferase, and 100 μm folate (or 10 μm dl-5-MeH₄folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B₁₂ enzyme content over the level already induced by 100 μm folate in 0.1 mm methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH₄folate fail to induce elevated amounts of B₁₂ methyltransferase in this mutant. Excess 10 mm medium methionine does, however, elevate its B₁₂ enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B₁₂ enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B₁₂ methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media.     

S-adenosylmethionine and morphogenesis inMucor racemosus

The intracellular concentration of S-adenosylmethionine (SAM) and the specific activity of S-adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6) were examined in wild-typeMucor racemosus, as well as a morphological mutant termedcoy, under conditions designed to prevent the morphogenesis of yeasts to hyphae. When the mutant was grown in a defined medium supplemented with methionine and induced to shift by exposure to air, there was an increase in intracellular SAM analogous to that previously reported with the wild type. However, when thecoy mutant was grown in the absence of methionine, the intracellular concentration decreased dramatically and the mutant failed to undergo the yeast to hypha transition. An inhibitor of SAM synthetase activity, cycloserine, was used to lower the intracellular concentration of SAM in the wild-type organisms. Under these conditions, wild-typeM. racemosus failed to undergo the transition from yeasts to hyphae when exposed to air.     

Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities

A morpholinepropanesulfonic acid (MOPS)-buffered rich defined medium (RDM) was optimized to support a reproducible 2.6-h doubling time at 35 °C for Deinococcus radiodurans R1 and used to gain insight into vitamin and carbon metabolism. D. radiodurans was shown to require biotin and niacin for growth in this medium. A glutamine–serine simple defined medium (SDM) was developed that supported a 4-h doubling time, and this medium was used to probe sulfur and methionine metabolism. Vitamin B₁₂ was shown to alleviate methionine auxotrophy, and under these conditions, sulfate was used as the sole sulfur source. Phenotypic characterization of a methionine synthase deletion mutant demonstrated that the B₁₂ alleviation of methionine auxotrophy was due to the necessity of the B₁₂-dependent methionine synthase in methionine biosynthesis. Growth on ammonium as the sole nitrogen source in the presence of vitamin B₁₂ was demonstrated, but it was not possible to achieve reproducibly good growth in the absence of at least one amino acid as a nitrogen source. Growth on sulfate, cysteine, and methionine as sulfur sources demonstrated the function of a complete sulfur recycling pathway in this strain. These studies have demonstrated that rapid growth of D. radiodurans R1 can be achieved in a MOPS-based medium solely containing a carbon source, salts, four vitamins, and two amino acids.     

Regulation of ornithine decarboxylase synthesis. Effect of a nutritional deficiency of vitamin B6

In vitamin B₆ deficiency there is an increase in the activity of the pyridoxal phosphate dependent enzyme ornithine decarboxylase. In the rat liver: the apoenzyme and holoenzyme activity increased 1.6 and 4 fold respectively. Concomitantly, putrescine and spermidine concentrations were halved. The lack of correspondence between product concentration and enzymic activity suggests a control mechanism other than ornithine decarboxylase activity.     

N5-methyltetrahydrofolate: Homocysteine methyltransferase activity in extracts from normal,malignant and embryonic tissue culture cells

Malignant cells (J111, L1210, W-256) and human embryonic cells (FL) are unable to survive and grow when homocystine replaces methionine in tissue culture media containing excess vitamin B₁₂ and folic acid. Extracts of these same cells when grown in media containing methionine and more than adequate vitamin B₁₂ and folic acid have diminished N⁵-methyltetrahydrofolate: homocysteine methyltransferase activities in the absence of added cyanocobalamin when compared with extracts of normal cells (adult rat thymus and liver fibroblasts). Extracts of human monocytic leukemia (J111) and human amnion cells (FL) have normal enzymatic activity in the presence of added cyanocobalamin whereas the rodent malignant cells (W-256 and L1210) have abnormally low activity in the absence or presence of added vitamin B₁₂.