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.     

Characterization of a vitamin B12 compound in the edible purple laver, Porphyra yezoensis

The edible purple laver, Porphyra yezoensis, contained 51.49+/-1.51 microg 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.     

Uptake and physiological function of vitamin B12 in a photosynthetic unicellular coccolithophorid alga, Pleurochrysis carterae

The photosynthetic coccolithophoid alga, Pleurochrysis (Hymenomonas) carterae, could take up and accumulate exogenous vitamin B12, most of which was converted into the coenzyme forms of vitamin B12. Two vitamin B12-dependent enzyme activities (methylmalonyl-CoA mutase, 2.6+/-0.4 nmol/min/mg protein and methionine synthase, 85.1+/-38.9 pmol/min/mg protein) could be found in a cell homogenate of the vitamin B12-supplemented alga. Most of the methylmalonyl-CoA mutase activity and 19.2% of the vitamin B12 accumulated by the algal cells were recovered in the macromolecular fractions with Mr of 150 kDa, although the remaining vitamin B12 was found only in free vitamin B12 fractions.     

Effect of nucleotide containing corrinoids on vitamin B 12 synthesis in Propionibacterium shermanii]

The influence of B12-CN, B12-OH, coenzyme B12, factor III and factor B on the synthesis of vitamin B12 and porphyrins by different strains of P. shermanii was investigated. Neither compound inhibited the development of propionic bacteria or suppressed porphyrin formation. All nucleotide containing analogues of vitamin B12 produced a strong repressive effect on the synthesis of corrinoid compounds regardless of the modifications in the upper and lower cobalt ligands. Factor B containing no nucleotide moiety did not show this effect. It is suggested that the nucleotide moiety of the vitamin B12 molecule is responsible for the binding of vitamin to protein aporepressor.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Cobinamides as structural probes in B12-biosynthesis: synthesis and spectroscopic analysis of isomers of Co alpha,Co beta-dicyanocobinamide

Vitamin B(12) and its coenzyme forms are cobalamins (i.e., cobamides, 'complete' with a 5,6-dimethylbenzimidazole nucleotide base), in which the particular corrinoid moiety of the cobinamides is conjugated to alpha-ribazole-3'-phosphate via a phosphate-diester group. Aside of being provided with their particular reactivity, required for their functions as organometallic cofactors in B(12)-dependent enzymes, the cobalamins also depend upon their specific three-dimensional buildup, to be able to adapt the unique constitution of 'base-on' corrinoids by intramolecular Co-coordination of the nucleotide base. We report rational partial syntheses and detailed spectral analyses of three close cobinamide isomers in their Co(alpha),Co(beta)-dicyano forms: of 13-epicobinamide (also called neocobinamide), of 176(S)-epicobinamide, and of 176-isocobinamide. Neocobinamide was obtained under acidic conditions as a degradation product of vitamin B(12). 176(S)-Epicobinamide and 176-isocobinamide were prepared by condensation of cobyric acid with (2S)-1-aminopropan-2-ol and with 3-aminopropan-1-ol, respectively. Natural cobinamide represents the corrinoid nucleus produced by proper microbial biosynthesis (as intermediate for the further assembly of the 'complete' corrinoid cofactors) or is required in some microorganisms, such as Escherichia coli, as an exogenously supplied unit for further biosynthetic buildup. The three compounds may thus be of use as structural probes for the biosynthetic capacity and tolerance in microorganisms, and (some of them) may serve as substrates as well, for further biosynthetic 'completion' of corrinoid cofactors or their analogues.     

Cobalamin-mediated mercury methylation by Desulfovibrio desulfuricans LS.

The prominence of sulfate reducers in mercury biomethylation prompted the examination of the methyl carrier and mercury methylation activity of Desulfovibrio desulfuricans LS. There was a low degree of mercury tolerance and a high degree of methylation during fermentative growth; the opposite was true during sulfate reduction. During 2 days of fermentative growth, up to 37% of HgCl2 was methylated at 0.1 micrograms/ml, but only 1.5% was methylated at 10.0 micrograms/ml. Less than 1% of the added HgCl2 was methylated under sulfate-reducing conditions. D. desulfuricans LS radioimmunoassay results were positive for cobalamin. The addition of CoCl2 and benzimidazole to fermentative cultures increased methylation activity. From D. desulfuricans LS grown in the presence of (57)CoCl2, a corrinoid was extracted and purified. High-performance liquid chromatography analysis of the purified extract yielded a single peak with the retention time of cobalamin, and 97% of the (57)Co radioactivity was associated with this peak. Fast atom bombardment and UV and visible spectra of the isolated corrinoid matched those of cobalamin. When methylated with (14)CH3I, the isolated corrinoid methylated Hg(2+) with a 93.9% preservation of (14)C specific activity. We conclude that D. desulfuricans LS methylates mercury via cobalamin (vitamin B12). Under physiological conditions, the enzymatic catalysis of this reaction is likely.     

Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans: substrate specificity of the native enzyme and its corrinoid cofactor

The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivoransand its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B(12)) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.     

Identification of phenolyl cobamide from the homoacetogenic bacterium Sporomusa ovata

Phenolyl cobamide was isolated from cyanide extractions of the anaerobic eubacterium Sporomusa ovata. The proposed corrinoid structure [Co alpha,Co beta-(monocyano,monoaquo)-phenolyl cobamide] has been deduced from 1H NMR, fast-atom-bombardment mass spectroscopy and ultraviolet/visible spectroscopy data. The complete corrinoid resembled p-cresolyl cobamide [Co alpha,Co beta-(monocyano,monoaquo)-p-cresolyl cobamide], which recently has been obtained from cyanide extractions of the same bacterium. The structures and chemical properties of both cobamides with uncoordinated nucleotides differed significantly from those of vitamin B12 [Co alpha-[alpha-(5,6-dimethylbenzimidazolyl)]-Co beta-cyanocobamide]. Sporomusa synthesized coenzymes of phenolyl cobamide and p-cresolyl cobamide in considerable amounts of 400 nmol/g and 1700 nmol/g dry cells, respectively. More than 90% of the complete corrinoid pool of the homoacetogenic bacterium consisted of these two corrinoids, indicating that they are physiologically important coenzymes of the bacterial metabolism.     

Isolation of a 5-hydroxybenzimidazolyl cobamide-containing enzyme involved in the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in Methanobacterium thermoautotrophicum.

Formaldehyde conversion into methyl-coenzyme M involves (a) reaction of the substrate with 5,6,7,8-tetrahydromethanopterin (H4MPT) giving 5,10-methylene-H4MPT, followed by its reduction to 5-methyl-H4MPT and (b) transfer of the methyl group from the latter compound to coenzyme M. The reactions were studied in a resolved system from Methanobacterium thermoautotrophicum strain delta H. The first part (a) of the reactions was catalyzed by the 55% ammonium sulfate supernatant of cell-free extracts. The methyltransferase step (b) was dependent on an oxygen-sensitive enzyme, called methyltransferase a (MTa). Isolation of MTa was achieved by gel filtration on Sephacryl S-400. MTa was a high-molecular-weight complex of at least 2000 kDa and between 900 to 1500 kDa when purified in the absence and presence of the detergent CHAPS, respectively. The enzyme consisted of 100 kDa units composed of three subunits in an alpha beta gamma configuration with apparent molecular masses of 35, 33 and 31 kDa, respectively. The corrinoid, 5-hydroxybenzymidazolyl cobamide (B12HBI, Factor III) copurified with MTa and the latter contained 2 nmol B12HBI per mg protein. B12HBI present in MTa could be methylated under the appropriate conditions by 5-methyl-H4MPT. These findings suggest that the corrinoid is a prosthetic group of MTa. MTa may be homologous to the corrinoid membrane protein purified before from M. thermoautotrophicum strain Marburg (Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Eur. J. Biochem. 171, 589-597).     

Fluorinated vitamin b(12) analogs are cofactors of corrinoid-dependent enzymes: a f-labeled nuclear magnetic resonance probe for identifying corrinoid-protein interactions

The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B(12) analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B(12). Hence, it is possible to use F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.     

The nitrogen-fixing symbiotic bacterium Mesorhizobium loti has and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B6

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway. The gene was cloned and ligated into pET-21a+. Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as K(m) values for pyridoxine (213+/-19 microM) and oxygen (78+/-10 microM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B6.     

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.     

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.     

A Salmonella typhimurium cobalamin-deficient mutant blocked in 1-amino-2-propanol synthesis.

Salmonella typhimurium synthesizes cobalamin (vitamin B12) when grown under anaerobic conditions. All but one of the biosynthetic genes (cob) are located in a single operon which includes genes required for the production of cobinamide and dimethylbenzimidazole, as well as the genes needed to form cobalamin from these precursors. We isolated strains carrying mutations (cobD) which are unlinked to any of the previously described B12 biosynthetic genes. Mutations in cobD are recessive and map at minute 14 of the linkage map, far from the major cluster of B12 genes at minute 41. The cobD mutants appear to be defective in the synthesis of 1-amino-2-propanol, because they can synthesize B12 when this compound is provided exogenously. Labeling studies in other organisms have shown that aminopropanol, derived from threonine, is the precursor of the chain linking dimethylbenzimidazole to the corrinoid ring of B12. Previously, a three-step pathway has been proposed for the synthesis of aminopropanol from threonine, including two enzymatic steps and a spontaneous nonenzymatic decarboxylation. We assayed the two enzymatic steps of the hypothetical pathway; cobD mutants are not defective in either. Furthermore, mutants blocked in one step of the proposed pathway continue to make B12. We conclude that the aminopropanol for B12 synthesis is not made by this pathway. Expression of a lac operon fused to the cobD promoter is unaffected by vitamin B12 or oxygen, both of which are known to repress the main cob operon, suggesting that the cobD gene is not regulated.     

Biosynthesis of corrinoids and other tetrapyrrole compounds by an acetogenic Clostridium

Biosynthesis of corrinoids and other tetrapyrrole pigments by the pure culture of the acetogenic Clostridium 99 was studied. When growing on media containing glucose or methanol, the physiological and biochemical characteristics of Clostridium 99 are very close to those of C. thermoautotrophicum. Methanol was shown to stimulate the corrinoid accumulation with the yield increasing from 154 micrograms/g dry biomass (glucose medium) up to 2250 micrograms/g dry biomass (methanol medium). According to the paper chromatography the corrinoid accumulated in Clostridium 99 cells differed both from vitamin B12 and Factor III. A study on the composition of extracellular tetrapyrroles, accumulated when the culture grows on the medium containing glucose and delta-aminolevulinic acid, revealed that they are represented both by uroporphyrin III and sirohydrochlorine-like pigments. The latters differ by a number of properties from sirohydrochlorine (corrifirine-2) of propione acidic bacteria. These pigments appear to be involved as intermediants in biosynthesis of corrinoids and other tetrapyrroles.     

Neovitamin B12 (cyano-13-epicobalamin).

Beta-Epimerization in the corrinoid system has recently emerged as a complicating factor in the latter stages of the total synthesis of vitamin B12 (Eschenmoser 1971; Woodward 1973). It has also found some application in biosynthetic studies on the origin of the methyl groups in ring C (Scott, Townsend & Cushley 1973; Scott, this Discussion p. 303). This paper sets out to review briefly the beta-epimerization of corrinoid polyamides, with particular reference to our work on the neo-series which provided the first established example of this phenomenon.     

Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.     

Molecular recognition in the binding of vitamin B12 by the cobalamin-specific Intrinsic Factor

Equilibrium constants (given as log K/M-1) have been determined at pH 7.4 and 4 degrees C for binding by porcine Intrinsic Factor (B12-binding protein from the gut, specific for the 'cobalamin' series of Co corrinoids) of vitamin B12 or cyanocobalamin (10.5), cyanocobinamide, alpha-ribazole and alpha-ribazole-phosphate (main fragments produced by cleaving off the 'cobalamin' side-chain, all less than or equal to 3), and cyanocobinamide in the presence of greater than or equal to 10(-9) M ribazole (5.6 and independent of ribazole concentration), i.e. ribazole catalyses the binding of the cobinamide. It is proposed that the specificity of Intrinsic Factor for the cobalamins depends on the presence of the ribazole fragment in the cobalamin side-chain to promote an essential change in conformation before the corrinoid fragment can be bound.     

Ribonucleotide reduction and the possible role of cobalamin in evolution

The biological pathways of ribonucleotide reduction are briefly reviewed. The hypothesis is presented that reduction of ribonucleoside triphosphates to their deoxynucleotide analogs through the mediation of vitamin B12 or a similar corrinoid preceded and was necessary for the subsequent development of a DNA-type genome. There are two known biological systems for ribonucleotide reduction: (1) The ribonucleoside diphosphate reduction system which utilizes a nonheme iron ribonucleotide reductase enzyme, thioredoxin and its reductase, and NADPH. This enzyme complex is found in most bacteria, some higher organisms, and in all animals. (2) The ribonucleoside triphosphate reduction system which utilizes adenosyl cobalamin, ribonucleotide reductase and either thioredoxin or a disulfhydryl compound. The cobalamin-dependent reductase is restricted to a few species of bacteria and blue-gree algae. This system is considered more primitive than the iron reductase one based on their differences in distribution, components, and products.