Vitamin B12-dependent Methionine Research Articles

Exploration of vitamin B12-producing bacteria from Indonesia Eutrophic Lake: A new strategy to improve microalgae biomass production

Abstract. Prabaningtyas S, Ardyati T, Suharjono, Retnaningdyah C. 2021. Exploration of vitamin B12-producing bacteria from Indonesia Eutrophic Lake: A new strategy to improve microalgae biomass production. Biodiversitas 22: 4538-4544. Biofuel producing autotrophs and heterotrophs organisms are mostly found in freshwaters such as lakes. Preliminary research showed that Ranu Grati has a high diversity of microalgae and bacteria. Microalgae are known as one source of biofuels due to the high content of lipid. The growth of microalgae is strongly influenced by vitamin B12, which is synthesized by bacteria. The role of vitamin B12 in the metabolism of algae is mainly as a co-enzyme for vitamin B12-dependent methionine synthase. The purpose of this research was to observe the bacterial diversity based on NGS analysis, to explore vitamin B12 produced by bacteria from Ranu Grati lake, and also to identify the highest potency of vitamin B12-producing bacteria based on 16S rRNA gene sequencing. The study was carried out by taking a water sample at a depth of 50 cm at five stations. Isolation of total bacterial DNA was carried out using FastDNA Spin Kit for Soil. The metagenomic method of Next Generation Sequencing (NGS) was used as an initial study of bacterial diversity. Vitamin B12-producing bacteria was isolated using PYBG agar (phytone peptone, trypticase peptone, lab-lemco powder, bacto yeast extract, glucose, agar). Isolated bacteria were screened for production of vitamin B12. The potential isolates were identified base on 16S rRNA gene sequence similarity. The results of the metagenomic study showed that the genus of potential bacteria producing vitamin B12 included Bacillus, Pseudomonas, Propionibacterium, Enterobacter, Escherichia, Acinetobacter, and Flavobacterium. The results of screening with PYBG media obtained 30 isolates of vitamin B12 producing bacteria. Ten efficient vitamin B12-producing isolates were identified as Lysinibacillus fusiformis (isolate G2V1), Bacillus cereus group (isolate G2V25, G2V24, G2V13, G2V9, G2V8), Alcaligenes faecalis (isolate G2V22), Delftia acidovorans (isolate G2V14A) and the genus Delftia (isolate G2V19). The isolate G2V1 (Lysinibacillus fusiformis) was the highest producer of vitamin B12 which was able to produce 33,783 ug/mL of vitamin B12 at 4 days incubation time.

Biological Activity of Pseudovitamin B12 on Cobalamin-Dependent Methylmalonyl-CoA Mutase and Methionine Synthase in Mammalian Cultured COS-7 Cells.

Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.

The effect of trichloroethylene metabolites on the hepatic vitamin B12-dependent methionine salvage pathway and its relevance to increased excretion of formic acid in the rat

The industrial solvent trichloroethylene (TCE) and its two major metabolites trichloroethanol (TCE-OH) and trichloroacetic acid (TCA) cause formic aciduria in male F344 rats. Prior treatment of male F344 rats with 1-aminobenzotriazole a cytochrome P450 inhibitor, followed by TCE (16mk/kg, po), completely prevented formic aciduria, but had no effect on formic acid excretion produced by TCA (8 or 16mg/kg, po), suggesting TCA may be the proximate metabolite producing this response. Dow and Green reported an increase in the concentration of 5-methyltetrahydrofolate (5-MTHF) in the plasma of rats treated with TCE-OH, suggesting a block in the cycling of 5-MTHF to tetrahydrofolate (THF). This pathway is under the control of the vitamin B12-dependent methionine salvage pathway. We therefore treated rats with three daily doses of methylcobalamin (CH3Cbl) or hydroxocobalamin (OHCbl), a cofactor for methionine synthase, or L-methionine, followed by TCE (16mg/kg) to determine if they could alleviate the formic aciduria. These pretreatments only partially reduced the excretion of formic acid in the urine. Although prior treatment with S-adenosyl-L-methionine had no effect on formic acid excretion. Consistent with these findings, the activity of methionine synthase in the liver of TCE-treated rats was not inhibited. Transcriptomic analysis of the liver-identified nine differential expressed genes, of note, was downregulation of Lmbrd1 involved in the conversion of vitamin B12 into CH3Cbl, a cofactor for methionine synthase. Our findings indicate that the formic aciduria produced by TCE-OH and TCA may be the result of a block in the recycling of 5-MTHF to THF, the effect on the methionine salvage pathway being a secondary response following acute exposure.

Laboratory assessment of folate (vitamin B9) status

Folate (vitamin B9) plays a crucial role in fundamental cellular processes, including nucleic acid biosynthesis, methyl group biogenesis and amino acid metabolism. The detection and correction of folate deficiency prevents...

Characterization and Quantitation of Vitamin B12 Compounds in Various Chlorella Supplements.

Vitamin B12 was determined and characterized in 19 dried Chlorella health supplements. Vitamin contents of dried Chlorella cells varied from <0.1 μg to approximately 415 μg per 100 g of dry weight. Subsequent liquid chromatography/electrospray ionization-tandem mass spectrometry analyses showed the presence of inactive corrinoid compounds, a cobalt-free corrinoid, and 5-methoxybenzimidazolyl cyanocobamide (factor IIIm) in four and three high vitamin B12-containing Chlorella tablets, respectively. In four Chlorella tablet types with high and moderate vitamin B12 contents, the coenzyme forms of vitamin B12 5'-deoxyadenosylcobalamin (approximately 32%) and methylcobalamin (approximately 8%) were considerably present, whereas the unnaturally occurring corrinoid cyanocobalamin was present at the lowest concentrations. The species Chlorella sorokiniana (formerly Chlorella pyrenoidosa) is commonly used in dietary supplements and did not show an absolute requirement of vitamin B12 for growth despite vitamin B12 uptake from the medium being observed. In further experiments, vitamin B12-dependent methylmalonyl-CoA mutase and methionine synthase activities were detected in cell homogenates. In particular, methionine synthase activity was significantly increased following the addition of vitamin B12 to the medium. These results suggest that vitamin B12 contents of Chlorella tablets reflect the presence of vitamin B12-generating organic ingredients in the medium or the concomitant growth of vitamin B12-synthesizing bacteria under open culture conditions.

Changes in Methionine Metabolism and Histone H3 Trimethylation Are Linked to Mitochondrial Defects in Multiple Sclerosis.

For decades, it has been observed that vitamin B12 deficiency and multiple sclerosis (MS) share certain pathological changes, including conduction disturbances. In the present study, we have found that vitamin B12-dependent methionine metabolism is dysregulated in the MS brain. We found that concentrations of the methyl donor betaine are decreased in MS cortex and are correlated with reduced levels of the histone H3 methyl mark H3K4me3 in neurons. Cell culture and chromatin immunoprecipitation-seq data suggest that these changes may lead to defects in mitochondria and impact neuronal energetics. These data have uncovered a novel pathway linking methionine metabolism with mitochondrial respiration and have important implications for understanding mechanisms involved in neurodegeneration in MS.

Betaine-Homocysteine Methyltransferase-2: cDNA Cloning, Gene Sequence, Physical Mapping, and Expression of the Human and Mouse Genes

Anomalies in folate and homocysteine metabolism can result in homocysteinemia and are implicated in disorders ranging from vascular disease to neural tube defects. Two enzymes are known to methylate homocysteine, vitamin B12-dependent methionine synthase (MTR) and betaine-homocysteine methyltransferase (BHMT). BHMT uses betaine, an intermediate of choline oxidation, as a methyl donor and is expressed primarily in the liver and kidney. We report the discovery of a novel betaine-homocysteine methyltransferase gene in humans and mice. The human BHMT2 gene is predicted to encode a 363-amino-acid protein (40.3 kDa) that shows 73% amino acid identity to BHMT. The BHMT2 transcript in humans is most abundant in adult liver and kidney and is found at reduced levels in the brain, heart, and skeletal muscle. The mouse Bhmt2 gene shows 69% amino acid identity and 79% similarity to the mouse Bhmt gene and 82% amino acid identity and 87% similarity to the human BHMT2 gene. Bhmt2 is expressed in fetal heart, lung, liver, kidney and eye. The discovery of a third gene with putative homocysteine methyltransferase activity is important for understanding the biochemical balance in using methyltetrahydrofolate and betaine as methyl donors as well as the metabolic flux between folate and choline metabolism in health and disease.

Purification, Kinetic Properties, and cDNA Cloning of Mammalian Betaine-Homocysteine Methyltransferase

Porcine liver betaine-homocysteine methyltransferase (BHMT; EC) was purified to homogeneity, and the Michaelis constants for betaine, dimethylacetothetin, and L-homocysteine are 23, 155, and 32 microM, respectively. The maximum rate of catalysis is 47-fold greater using dimethylacetothetin as a methyl donor compared with betaine. Partial amino acid sequence of porcine BHMT was obtained, and inosine-containing redundant oligonucleotide primers were used to amplify an 815-base pair sequence of the porcine cDNA by polymerase chain reaction (PCR). Nondegenerate oligonucleotide primers based on the porcine cDNA were synthesized and used to isolate a 463-base pair fragment of the human cDNA by PCR. The human PCR DNA product was then used to screen a cDNA library by plaque hybridization, and cDNAs encoding human BHMT were isolated. The primary structure of the human cDNA is reported here, and the open reading frame encodes a 406-residue protein of Mr 44,969. The deduced amino acid sequence of human BHMT shows limited homology to bacterial vitamin B12-dependent methionine synthases (EC). A plasmid containing the human BHMT cDNA fused in frame to the N terminus of beta-galactosidase was transformed into Escherichia coli, and transformants expressed BHMT activity, an activity that is absent from wild type E. coli.

Stimulation in vitro of vitamin B12-dependent methionine synthase by polyamines.

Vitamin B12-dependent methionine synthase is an important enzyme for sulphur amino acid, folate polyamine metabolism, S-adenosylmethionine metabolism and also in the methylation pathway of DNA, RNA, proteins and lipids. Consequently, studies aiming at exploring the control and regulation of methionine synthase are of particular interest. Here we report the modulation of enzyme activity in vitro by polyamines. Although putrescine, cadaverine, spermine and spermidine all stimulated enzyme activity, the last two were the most potent, causing increases in enzyme activity up to 400%. The EC50 for spermine was determined as 8 microM and for spermidine 40 microM. The physiological concentration for spermine has been reported to be 15-19 microM. Spermine was found to increase both the Km and the V(max) with respect to methyltetrahydrofolate for the enzyme. These data support the hypothesis that spermine and spermidine are feedback regulators of methionine synthase both in vivo and in vitro and are consistent with the polyamines' regulating cell signalling pathways.

Sulphur amino acid derivatives as inhibitors of vitamin B12 dependent methionine synthase.

Sulphur amino acid derivatives as inhibitors of vitamin B12 dependent methionine synthase.

Mutations in the B12-binding region of methionine synthase: how the protein controls methylcobalamin reactivity.

Vitamin B12-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine via the enzyme-bound cofactor methylcobalamin. To carry out this reaction, the enzyme must alternately stabilize six-coordinate methylcobalamin and four-coordinate cob(I)alamin oxidation states. The lower axial ligand to the cobalt in free methylcobalamin is the dimethylbenzimidazole nucleotide substituent of the corrin ring; when methylcobalamin binds to methionine synthase, the ligand is replaced by histidine 759, which in turn is linked by hydrogen bonds to aspartate 757 and thence to serine 810. We have proposed that these residues control the reactivity of the enzyme-bound cofactor both by increasing the coordination strength of the imidazole ligand and by allowing stabilization of cob(I)alamin via protonation of the His-Asp-Ser triad. In this paper we report results of mutation studies focusing on these catalytic residues. We have used visible absorbance spectroscopy and electron paramagnetic resonance spectroscopy to probe the coordination state of the cofactor and have used stopped-flow kinetic measurements to explore the reactivity of each mutant. We show that mutation of histidine 759 blocks turnover, while mutations of aspartate 757 or serine 810 decrease the reactivity of the methylcobalamin cofactor. In contrast, we show that mutations of these same residues increase the rate of AdoMet-dependent reactivation of cob(II)alamin enzyme. We propose that the reaction with AdoMet proceeds via a different transition state than the reactions with homocysteine and methyltetrahydrofolate. These results provide a glimpse at how a protein can control the reactivity of methylcobalamin.

Polyamines can regulate vitamin B12 dependent methionine synthase activity.

Polyamines can regulate vitamin B12 dependent methionine synthase activity.

In vitro NO and N2O inhibition of the branch point enzyme vitamin B12 dependent methionine synthase from rat brain synaptosomes.

In vitro NO and N2O inhibition of the branch point enzyme vitamin B12 dependent methionine synthase from rat brain synaptosomes.

Nitric oxide effects on polyamine pathways in cultured hepatocytes.

0 Vitamin B12dependent methionine synthase has been shown to be inhibited by nitric oxide in a time dependent and dose-dependent manner in vitro [ 11. This enzyme is the first step in decarboxy Sadenosyl methionine (dcSAM) formation and hence should affect polyamine levels in cells. It is important therefore, in correlating in vitro and cellular effects of NO to establish in a single cell system that NO affects those metabolic pathways that use dcSAM as cofactor (see Fig. 1). HCys MET SAM dcSAM

Effect of nitrous oxide on nickel deprivation in rats.

Because nickel may have a biological function in a pathway in which vitamin B12 is important, an experiment was performed to determine the effects of nitrous oxide exposure in rats deprived of nickel. Exposure to nitrous oxide (N2O) causes inactivation of cobalamin and a subsequent decrease in the vitamin B12-dependent enzymes methionine synthase and methylmalonyl CoA mutase. Rats were assigned to dietary groups of 12 in a factorially arranged experiment with dietary variables of nickel (0 or 1 microgram/g) and vitamin B12 (0 or 50 ng/g). After 6 wk, one-half of the rats from each dietary group were exposed to 50% N2O/50% O2 for 90 min/d for the last 28 d of the experiment. Vitamin B12, N2O, or their interaction had numerous effects; classical findings included N2O-induced reduction in plasma vitamin B12 and decreases in the vitamin B12-dependent enzymes. Inactivation of vitamin B12 by N2O, however, did not exacerbate signs of nickel deprivation, possibly because the rats were able to metabolically compensate to N2O exposure.

Dietary vitamin B12, sulfur amino acids, and odd-chain fatty acids affect the response of rats to nickel deprivation

An experiment was performed to ascertain whether changing the dietary intake of two substances, cystine and margaric acid (heptadecanoic acid), that affect the flux through pathways involving the two vitamin B12-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase, would affect the interaction between nickel and vitamin B12. Rats were assigned to treatment groups of six in a fully crossed, four-factorial arrangement. The independent variables, or factors, were: per kg of fresh diet, nickel analyzed at 25 and 850 micrograms; vitamin B12 supplements of 0 and 50 micrograms; margaric acid supplements of 0 and 5 g; and L-cystine supplements of 0 and 12 g. The diet without cystine was marginally deficient in sulfur amino acids. Nickel affected growth, liver wt/body wt ratio (LB/BW), and a number of variables associated with iron, calcium, zinc, copper, and magnesium metabolism. Most of the effects of nickel were modified by the vitamin B12 status of the rat. In numerous cases, the interaction between nickel and vitamin B12 was dependent on, or altered by, the cystine or margaric acid content of the diet. Thus, the findings showed that the extent and the direction of changes in numerous variables in response to nickel deprivation varied greatly with changes in diet composition. These variables include those previously reported to be affected by nickel deprivation, including growth and the distribution or functioning of iron, calcium, zinc, copper, and magnesium. The findings also support the hypothesis that nickel has a biological function in a metabolic pathway in which vitamin B12 is important.

Evidence of brain methyltransferase inhibition and early brain involvement in HIV-positive patients

The myelopathy associated with human immunodeficiency virus (HIV) infection closely resembles that in subacute combined degeneration, a disorder of vitamin B 12 metabolism. To investigate whether the disorders share a pathogenetic mechanism, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were measured in the cerebrospinal fluid (CSF) of 20 HIV-seropositive patients and 30 HIV-negative patients who were undergoing lumbar puncture for other medical reasons. The HIV-seropositive patients had significantly lower CSF concentrations of SAM (mean 77 [SD 25] vs 131 [35] nmol/l; p < 0·001) and significantly higher concentrations of SAH (30·5 [6·8] vs 19·0 [7·1] nmol/l; p < 0·001) than the controls. There was therefore a significant difference between the groups in the SAM/SAH (methylation) ratio (HIV 2·7 [1·0] vs control 7·6 [3·4]; p < 0·001). There were no correlations between SAM or SAH concentrations or methylation ratio and age or sex in both groups, or serum B 12 and folate concentrations, CSF folate, serum or CSF methylmalonic acid, risk factors, body mass index, specific drug treatment received, or disease stage in the HIV group. This finding suggests that HIV affects the brain from a very early stage of the infection. We suggest that, as in the pig, the CSF methylation ratio closely reflects that in the brain. In HIV-infected patients a reduced brain methylation ratio would inhibit methyltransferase enzymes, which would lead to hypomethylation in the central nervous system and ultimately to neurological lesions. In a pig model of subacute combined degeneration and in vitamin-B 12-deficient human beings, the primary cause of the low methylation ratio is impaired recycling of SAH back to SAM, a process which requires vitamin-B 12-dependent methionine synthase. The HIV patients in this study were vitamin B 12 and folate replete, which suggests a different cause for the low methylation ratio.

Methylmalonyl-CoA mutase (EC5.4.99.2) and methionine synthetase (EC2.1.1.13) in the tissues of cobalt–vitamin B12deficient sheep

The changes in the activities of the two vitamin B12-dependent enzymes methylmalonyl-CoA mutase (EC 5.4.99.2) and methionine synthetase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) are described in two groups of sheep maintained for 20 weeks on either a cobalt-deficient or a Co-sufficient whole-barley diet. At the end of that period, the plasma concentrations of vitamin B12 were depressed and those of methylmalonic acid were raised in the Co-deficient group. During the course of the experiment hepatic holo-mutase activity, measured on biopsy samples, declined in Co-deficient animals with a half-life of 73 d. There was a similar, but slower decline in lymphocyte holo-mutase activity which fell with a half-life of 125 d. At slaughter, there was no difference between Co-sufficient and Co-deficient animals in total mutase activity in liver, kidney, brain and spinal cord. In contrast, the total-synthetase activity of liver and kidney was reduced by 60 and 30% respectively in the Co-deficient animals. There was no change in either group of animals in total-synthetase activity, or in either holo-mutase or holo-synthetase activity, in brain and spinal cord. In the Co-deficient animals, holo-mutase and holo-synthetase activities in liver, the tissue with the greatest activity of both enzymes, fell to 25 and 39% respectively, of that of Co-sufficient animals. The corresponding reductions for kidney were 12 and 51% respectively. These results indicated that activity of both holoenzymes is greatly reduced in Co-deficient sheep.

Plasma amino acids and tissue methionine levels in fruit bats (Rousettus aegyptiacus) with nitrous oxide-induced vitamin B12 deficiency.

The effect of methylcobalamin inactivation by the gas nitrous oxide on plasma amino acid and tissue methionine levels in fruit bats (Rousettus aegyptiacus) was examined. Animals exposed to N2O-oxygen (1:1, v/v) for 90 min daily received a fruit diet with or without methionine or betaine supplements. Exposure and diets were continued for up to 17 weeks or until neurological impairment and muscular weakness was established. All the groups exposed to N2O had significantly lower liver, brain and plasma methionine concentrations except the methionine-supplemented animals which showed significantly raised levels. Plasma homocysteine, which was absent in controls, was present in all the N2O-exposed groups. Betaine supplementation resulted in reduced accumulation of homocysteine in plasma. However, plasma and liver methionine levels were only slightly increased compared with animals on the basal diet, and brain methionine levels were the lowest of all the groups studied. These results support the hypothesis that reduced methionine synthesis is an important contributor to the development of neurological impairment in this species and suggest that dietary supplementation with the methionine precursor betaine cannot replace the loss of vitamin B12-dependent methionine synthesis.

Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis.

Cultured fibroblasts from a recently described patient with homocystinuria and megaloblastic anemia of infancy without methylmalonic aciduria were previously shown to have normal cobalamin uptake and a specific decrease in the proportion of intracellular methylcobalamin. As in control cells but unlike in those from patients with combined homocystinuria and methylmalonic aciduria (cobalamin C and cobalamin D), accumulated 57Co-labeled cobalamin was bound in appropriate amounts and proportion to intracellular binders which are known to be the two vitamin B12-dependent enzymes, methionine synthetase and methylmalonyl-CoA mutase. Despite the association of a normal quantity of intracellular cobalamin with methionine synthetase, the proportion of intracellular cobalamin which was methyl-B12 was below normal and in the range observed in cobalamin C and D cells. This methyl-B12 was decreased by exposure of fibroblasts in culture to nitrous oxide as was observed with control cells. Exposure of control fibroblasts during culture, but not of fibroblasts from this patient, to nitrous oxide significantly reduced the holoenzyme activity of methionine synthetase assayed in cell extracts. In addition, although methionine synthetase activity in cell extracts of control and cells from the patient were similar in the presence of standard assay concentrations of thiols, at low thiol concentrations, methionine synthetase activity in extracts of cells from the patient was much lower than in control extracts. Mixing of control patient extracts corrected this decreased activity in excess of that explained by addition of the individual activities added. The defect of this patient appears to be in a reducing system required for methionine synthesis.