Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety

In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1′-¹⁴C]riboflavin. The vitamin B₁₂ isolated was ¹⁴C-labeled. It was shown by chemical degradation that the ¹⁴C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium. Received: 25 August 1998 / Accepted: 27 October 1998     

Microbiological activities of nucleotide loop-modified analogues of vitamin B12

Novel vitamin B₁₂ analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B₁₂- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5.6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B₁₂ activity and behaved as weak B₁₂ antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. ON the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [³H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B₁₂ activity toward E. coli 215 by blocking the B₁₂-transport systemAbbreviations AdoCbl adenosylcobalamin - MeCbl methylcobalamin - CN-Cbl cyanocobalamin or vitamin B₁₂ - Cbl cobalamin - (CN, aq)Cbi cyanoaquacobinamide - MeCbi methylcobinamide - Cbi cobinamide - (CN, aq)Cbi-PMe cyanoaquacobinamide methyl phosphate - Cbi-PMe cobinamide methyl phosphate - DBI 5,6-dimethylbenzimidazole - DBIyl 5,6-dimethylbenzimidazolyl - FMNH₂ fully reduced form of riboflavin 5-phosphate     

Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii

1. Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethylbenzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. 2. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. 3. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3--0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. 4. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1'-14C]riboflavin as substrate the specific radioactivity of 5,6-dimethylbenzimidazole is higher than the specific radioactivity of flavin--adenine dinucleotide and lower than the specific radioactivie substrate for the formation of 5,6-dimethylbenzimidazole. 5. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed.     

Biosynthesis of vitamin B12. Transformation of riboflavin 2H-labeled in the 1'R position of 1'S position into 5,6-dimethylbenzimidazole.

The 5,6-dimethylbenzimidazole moiety of vitamin B12 is formed from riboflavin in aerobic and some aerotolerant bacteria. Thereby C1' of riboflavin is transformed into C2 of the vitamin B12 base. In the present publication a study on this transformation with riboflavin 2H-labeled in the 1'R or 1'S position is described. This study was undertaken in order to find out if one of the two hydrogens at C1' is transferred to C2 of 5,6-dimethylbenzimidazole. The 2H-labeled riboflavin samples were synthesized starting from unlabeled or 1-2H-labeled ribose and 3,4-dimethylaniline yielding N-beta-D-ribopyranosyl-3,4-dimethylaniline. The unlabeled riboside was reduced to N-D-ribityl-3,4-dimethylaniline with sodium cyanoborotrideuteride, the 2H-labeled riboside with sodium cyanoborohydride. The ribityl derivatives were transformed into N-D-ribityl-2-phenylazo-4,5-dimethylaniline, and condensed with barbituric acid to riboflavin. The reduction of the ribosyl compound to the ribityl derivative is only partially stereospecific. Thus the riboflavin synthesized from unlabeled ribose had a 2H ratio of 3/1 (1'R/1'S), the riboflavin obtained from D-[1-2H1]ribose of 1/3 (1'R/1'S). The 2H content in these positions was determined from the 1H-NMR spectra. These spectra showed also that 1 mol 2H/mol riboflavin was present in position 1'. The deuterated riboflavin samples were incubated under aerobic conditions with broken cell preparations of Propionibacterium shermanii. The deuterium content of the 5,6-dimethylbenzimidazole isolated was determined by mass spectrometry and by 1H NMR. These measurements revealed that the hydrogen in the pro-S position at C1' of riboflavin is retained during 5,6-dimethylbenzimidazole formation, and is thus found at C2 of this base.     

Effect of a corrinoid on <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> DNA synthesis

Methanosarcina barkeri is capable of synthesizing large amounts of corrinoids, compounds of the vitamin B₁₂ group, although not cobalamin. In the present work, exogenous cobalamin was demonstrated to upregulate DNA synthesis in M. barkeri cell suspensions incubated under air. The effect is similar to the one in Propionibacterium freudenreichii cells, though less pronounced. The growth of the archaeon under anaerobic conditions was shown to be suppressed by cobalamin and 5,6-dimethylbenzimidazole. The data obtained suggest the presence of a corrinoid-dependent ribonucleotide reductase in the archaeal cells which provides for deoxyribose precursors for DNA biosynthesis independently of the presence of molecular oxygen in the medium. Growth suppression under anoxic conditions by cobalamin and 5,6-dimethylbenzimidazole may be due to a decrease in the concentration of factor III, a polyfunctional corrinoid dominating in M. barkeri cells.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.     

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions.

In this paper, we report that the enteric bacterium Salmonella typhimurium synthesized cobalamin de novo under anaerobic culture conditions. Aerobically, metE mutants of S. typhimurium need either methionine or cobalamin as a nutritional supplement for growth. The growth response to cobalamin depends upon a cobalamin-requiring enzyme, encoded by the gene metH, that catalyzes the same reaction as the metE enzyme. Anaerobically, metE mutants grew without any nutritional supplements; the metH enzyme functioned under these conditions due to the endogenous biosynthesis of cobalamin. This conclusion was confirmed by using a radiochemical assay to measure cobalamin production. Insertion mutants defective in cobalamin biosynthesis (designated cob) were isolated in the three major branches of the cobalamin biosynthetic pathway. Type I mutations blocked the synthesis of cobinamide, type II mutations blocked the synthesis of 5,6-dimethylbenzimidazole, and type III mutations blocked the synthesis of cobalamin from cobinamide and 5,6-dimethylbanzimidazole. Mutants that did not synthesize siroheme (cysG) were blocked in cobalamin synthesis. Genetic mapping experiments showed that the cob mutations are clustered in the region of the S. typhimurium chromosome between supD (40 map units) and his (42 map units). The discovery that S. typhimurium synthesizes cobalamin de novo only under anaerobic conditions raises the possibility that anaerobically grown cells possess a variety of enzymes which are dependent upon cobalamin as a cofactor.     

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.     

Sirtuin chemical mechanisms

Sirtuins are ancient proteins widely distributed in all lifeforms of earth. These proteins are universally able to bind NAD⁺, and activate it to effect ADP-ribosylation of cellular nucleophiles. The most commonly observed sirtuin reaction is the ADP-ribosylation of acetyllysine, which leads to NAD⁺-dependent deacetylation. Other types of ADP-ribosylation have also been observed, including protein ADP-ribosylation, NAD⁺ solvolysis and ADP-ribosyltransfer to 5,6-dimethylbenzimidazole, a reaction involved in eubacterial cobalamin biosynthesis. This review broadly surveys the chemistries and chemical mechanisms of these enzymes.     

Guanylcobamide and hypoxanthylcobamide-Corrinoids formed by Desulfovibrio vulgaris

The corrinoids synthesized by the sulfate-reducing bacterium Desulfovibrio vulgaris were analyzed. The compounds found were guanylcobamide and hypoxanthylcobamide; structures were determined by mass spectrometry, ¹H-NMR, and ultraviolet/visible spectroscopy. D. vulgaris used externally added guanine to form guanylcobamide, as demonstrated with 8-¹⁴C-guanine. Addition of adenine did not lead to the formation of adenylcobamide (pseudovitamin B₁₂), whereas 5,6-dimethylbenzimidazole was transformed into vitamin B₁₂.     

Salmonella typhimurium A1-R targeting of a chemotherapy-resistant BRAF-V600E melanoma in a patient-derived orthotopic xenograft (PDOX) model is enhanced in combination with either vemurafenib or temozolomide

A metastatic melanoma obtained from the right chest wall of a patient was previously established orthotopically in the right chest wall of nude mice as a patient-derived orthotopic xenograft (PDOX) model. We previously showed that the combination of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and chemotherapy was highly effective against the melanoma PDOX. In the present study, we investigated the mechanism of the high efficacy of this combination. Two weeks after implantation, 40 PDOX mouse models were randomized into 4 groups of 10 mice each: untreated control (n = 10); treated with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with temozolomide (TEM) (25 mg/kg, p.o. for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with vemurafenib (VEM) (30 mg/kg, p.o., for 14 consecutive days) combined with S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks) (n = 10). On day 14 from initiation, all treatments significantly inhibited tumor growth compared with untreated control (S. typhimurium A1-R: p < 0.01; TEM combined with S. typhimurium A1-R: p < 0.01; VEM combined with S. typhimurium A1-R: p < 0.01). Combination therapy with S. typhimurium A1-R was significantly more effective on tumor growth than S. typhimurium A1-R alone (with TEM: p < 0.01; with VEM: p < 0.01). Combination therapy significantly increased S. typhimurium A1-R tumor targeting alone (S. typhimurium A1-R + TEM: p < 0.01, S. typhimurium A1-R + VEM: p < 0.01), relative to S. typhimurium A1-R alone, respectively. In conclusion, chemotherapy drugs promoted targeting of S. typhimurium A1-R of melanoma, thereby enhancing efficacy against the melanoma PDOX.     

Riboflavin status modifies the effects of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms on homocysteine

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), riboflavin-dependent enzymes, participate in homocysteine metabolism. Reported effects of riboflavin status on the association between the MTHFR 677C>T polymorphism and homocysteine vary, and the effects of the MTRR 66A>G or MTRR 524C>T polymorphisms on homocysteine are unclear. We tested the hypothesis that the effects of the MTHFR 677C>T, MTRR 66A>G and MTRR 524C>T polymorphisms on fasting plasma total homocysteine (tHcy) depend on riboflavin status (erythrocyte glutathionine reductase activation coefficient, optimum: <1.2; marginally deficient: 1.2–1.4; deficient: ≥1.4) in 771 adults aged 18–75 years. MTHFR 677T allele carriers with middle or low tertile plasma folate (<14.7 nmol/L) had 8.2 % higher tHcy compared to the 677CC genotype (p < 0.01). This effect was eliminated when riboflavin status was optimal (p for interaction: 0.048). In the lowest cobalamin quartile (≤273 pmol/L), riboflavin status modifies the relationship between the MTRR 66 A>G polymorphism and tHcy (p for interaction: 0.034). tHcy was 6.6 % higher in MTRR 66G allele carriers compared to the 66AA genotype with marginally deficient or optimal riboflavin status, but there was no difference when riboflavin status was deficient (p for interaction: 0.059). tHcy was 13.7 % higher in MTRR 524T allele carriers compared to the 524CC genotype when cobalamin status was low (p < 0.01), but no difference was observed when we stratified by riboflavin status. The effect of the MTHFR 677C>T polymorphism on tHcy depends on riboflavin status, that of the MTRR 66A>G polymorphism on cobalamin and riboflavin status and that of the MTRR 524C>T polymorphism on cobalamin status.     

Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide: 5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrates, giving rise to a wide variety of lower ligands in cobamides. To understand the molecular basis for this lack of substrate specificity, the x-ray structures of CobT complexed with adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, p-cresol, and phenol were determined. Furthermore, adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, and 2-hydroxypurine were observed to react with NaMN within the crystal lattice and undergo the phosphoribosyl transfer reaction to form product. Significantly, the stereochemistries of all products are identical to those found in vivo. Interestingly, p-cresol and phenol, which are the lower ligand in Sporomusa ovata, bound to CobT but did not react with NaMN. This study provides a structural explanation for how CobT can phosphoribosylate most of the commonly observed lower ligands found in cobamides with the exception of the phenolic lower ligands observed in S. ovata. This is accomplished with minor conformational changes in the side chains that constitute the 5,6-dimethylbenzimidazole binding site. These investigations are consistent with the implication that the nature of the lower ligand is controlled by metabolic factors rather by the specificity of the phosphoribosyltransferase.     

Analysis of the nucleoside moiety of cobalamin and cobalamin analogues using gas chromatography-mass spectrometry.

Existing techniques for identification of cobalamin and cobalamin analogues generally use the intact molecule during characterization with somewhat ambiguous results. In this study a method is described for the identification of the nucleoside in the lower axial ligand of cobalamin and a variety of naturally occurring cobalamin analogues that differ from cobalamin in the base that is present in the nucleoside. Cobalamin and cobalamin analogues were isolated from biological samples by affinity chromatography using R-protein-Sepharose columns. The nucleosides of the lower axial ligand were then hydrolyzed and isolated by column chromatography using a mixed bed column. Nucleosides were oxidized with periodate and reduced with borohydride. After reisolation, the t-butyldimethylsilyl derivatives were prepared and analyzed using gas chromatography/mass spectrometry with selected ion monitoring. A stable isotope internal standard of cobalamin was biosynthetically produced and used to quantitate cobalamin in rabbit kidney. Cobalamin analogues were also shown to be present in rabbit kidney, but they contain the 5,6-dimethylbenzimidazole nucleoside (alpha-ribazole) in the lower axial ligand, indicating that these analogues differ from cobalamin in the corrin ring region of the molecule.     

Cloning and Characterization of the Gene Encoding O-Acetylserine Lyase from Streptococcus suis

We have cloned and sequenced a gene encoding O-acetylserine lyase from Streptococcus suis. The gene encodes a protein of 309 amino acids with a calculated molecular mass of 32,038 Da. The deduced amino acid sequence showed more extensive similarities to the CysK proteins than to the CysM proteins of other bacteria. The cloned gene was inserted into a pTrcHisB histidine hexamer expression vector. A 38-kDa fusion protein was expressed in a cysMK auxotrophic mutant of Salmonella typhimurium and complemented the auxotrophic properties of the mutant. Furthermore, the transformants could grow in minimal defined media supplemented with not only sulfide but also thiosulfate as a sole sulfur source. These data indicated that the cloned gene encodes a protein that was a functional homolog of the CysM in S. typhimurium. Received: 1 July 1999 / Accepted: 10 August 1999     

The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin.

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) that catalyzes the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, a biosynthetic intermediate of the pathway that assembles the nucleotide loop of cobalamin in Salmonella typhimurium. Mutants previously isolated as DMB auxotrophs are shown by physical and genetic mapping studies and complementation studies to carry lesions in cobT. Explanations for this unexpected phenotype of cobT mutants are discussed. The expected nucleotide loop assembly phenotype of cobT mutants can be observed only in a specific genetic background, i.e., cobB deficient, an observation that is consistent with the existence of an alternative CobT function (G. A. O'Toole, M. R. Rondon, and J. C. Escalante-Semerena, J. Bacteriol. 175:3317-3326, 1993). Computer analysis of CobT homologs showed that at the amino acid level, enteric CobT proteins were 80% identical whereas Pseudomonas denitrificans and Rhizobium meliloti CobT proteins were 95% identical. Interestingly, the degree of identity between enteric and nonenteric CobT homologs was only 30%. The same pattern of homologies was reported for the S. typhimurium CobA, Escherichia coli BtuR, and P. denitrificans CobO proteins (S.-J. Suh and J.C. Escalante-Semerena, Gene 129:93-97, 1993), suggesting evolutionary divergence between the cob genes found in the enteric bacteria E. coli and S. typhimurium and those found in P. denitrificans and R. meliloti.     

The kinetics and mechanism of cobalamin-dependent methyl and ethyl transfer to mercuric ion

The chemical transfer of alkyl groups from alkylcobalamins to mercuric ion has been studied in detail by using ultraviolet-visible conventional and stopped-flow kinetics and, in the case of methyl group transfer, by 220 MHz NMR spectroscopy. These experiments show that heterolytic cleavage of the Co–C δ-bond occurs during electrophilic attack by mercuric ion to give alkylmercury and aquocobalamin as the reaction products. Equilibrium and kinetic experiments are consistent with the initial displacement of 5,6-dimethylbenzimidazole by mercuric ion which results in a deactivaion toward dealkylation by a second mercuric ion. Consequently the main dealkylation reaction at pH 5.0 occurs with uncomplexed alkylcobalamin with the overall rate k_obd being controlled by the above equilibrium. Both the displacement of 5,6-dimethylbenzimidazole (“fast reaction”) and dealkylation (“slow reaction”) are first order in the active mercuric species.     

Methanol conversion in Eubacterium limosum

The conversion of methanol by cell-free extracts of the acetogenic bacterium Eubacterium limosum was studied. Incubation of mixed cell-free extracts of both E. limosum and Methanobacterium formicicum resulted in methane formation from methanol in the presence of ATP and 2-mercaptoethanesulfonic acid. The separate extracts were not able to perform this reaction. Addition of ferredoxin obtained from Methanosarcina barkeri to the mixed extracts resulted in increased methane formation. The enzyme, responsible for methanol binding in cell-free extract of E. limosum, was inactivated by FAD under N₂ and exhibited maximal activity under an atmosphere of H₂. This enzyme contains a firmly bound cobalamin which was methylated by methanol in the presence of ATP. It was demethylated in the presence of methylcobalamin: coenzyme M methyltransferase obtained from M. barkeri under concomitant formation of methylated coenzyme M. These properties are similar to those of methanol: 5-hydroxybenzimidazolylcobamide methyltransferase from M. barkeri. It was proposed that methylotrophic acetogens and methylotrophic methanogens use similar enzymes in the first step of methanol conversion.Abbreviations HS-CoM 2-mercaptoethanesulfonic acid - CH₃S-CoM 2-(methylthio)ethanesulfonic acid - BrES 2-bromoethanesulfonic acid - TES N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid - MT₁ methanol: 5-hydroxybenzimidazolylcobamide methyltransferase - MT₂ methylcobalamin - HS-CoM methyltransferase - DMBI 5,6-dimethylbenzimidazole and HBI, 5-hydroxybenzimidazole, are -ligands of corrinoids - (S-CoM)₂ 2,2-dithiodiethanesulfonic acid