Vitamin B12-dependent Methionine Synthase 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.

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...

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.

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

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.