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PubMed2001

Characterization of a three-component vanillate O-demethylase from Moorella thermoacetica.

The Moorella thermoacetica aromatic O-demethylase was characterized as an inducible three-component system with similarity to the methanogenic methanol, methylamine, and methanethiol methyltransferases and to the O-demethylase system from Acetobacterium dehalogenans. MtvB catalyzes methyl transfer from a phenylmethylether to the cobalt center of MtvC, a corrinoid protein. MtvA catalyzes transmethylation from MtvC to tetrahydrofolate, forming methyltetrahydrofolate. Cobalamin can substitute for MtvC.

Amino Acid Sequence↗
PubMed2001

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.

Adenosine↗
PubMed2003

The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis.

Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain DeltaH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea. Computer analysis of the Halobacterium sp. strain NRC-1 ORF Vng1581C gene and the cobY gene of M. thermoautotrophicum strain DeltaH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar. A strain of Halobacterium sp. strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis. The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon. The data also show that Halobacterium sp. strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions. To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea.

Archaeal Proteins↗
PubMed2005

Effects of nitrogen and carbon sources on transcription of soluble methyltransferases in Methanosarcina mazei strain Go1.

The methanogenic archaeon Methanosarcina mazei strain Gö1 uses versatile carbon sources and is able to fix molecular nitrogen with methanol as carbon and energy sources. Here, we demonstrate that when growing on trimethylamine (TMA), nitrogen fixation does not occur, indicating that ammonium released during TMA degradation is sufficient to serve as a nitrogen source and represses nif gene induction. We further report on the transcriptional regulation of soluble methyltransferases, which catalyze the initial step of methylamine consumption by methanogenesis, in response to different carbon and nitrogen sources. Unexpectedly, we obtained conclusive evidence that transcription of the mtmB2C2 operon, encoding a monomethylamine (MMA) methyltransferase and its corresponding corrinoid protein, is highly increased under nitrogen limitation when methanol serves as a carbon source. In contrast, transcription of the homologous mtmB1C1 operon is not affected by the nitrogen source but appears to be increased when TMA is the sole carbon and energy source. In general, transcription of operons encoding dimethylamine (DMA) and TMA methyltransferases and methylcobalamine:coenzyme M methyltransferases is not regulated in response to the nitrogen source. However, in all cases transcription of one of the homologous operons or genes is increased by TMA or its degradation products DMA and MMA.

Archaeal Proteins↗
PubMed2006

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1.

Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Gö1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes.

Alkyl and Aryl Transferases↗
PubMed1992

Radioimmunoassay that measures serum vitamin B12.

AIMS: To develop a specific radioimmunoassay for the routine determination of serum vitamin B12. METHODS: Antisera were raised in rabbits by immunisation with the monocarboxylic acid derivative of cyanocobalamin coupled to human serum albumin. Antibody titres and affinities were determined and the antiserum giving the highest binding affinity constant, Ka, was used to develop the assay protocol. Donkey-anti-rabbit gamma globulin-coated magnetisable particles were used to separate the bound from free vitamin B12. The considerable cobalamin binding capacity of human serum was destroyed by autoclaving in acetate-cyanide buffer. Sixty samples were assayed by the radioimmunoassay (RIA) and the Lactobacillus leichmannii assay. Recovery and cross-reactivity experiments were performed. RESULTS: Final rabbit antibody titres varied from 1/20,000 to 1/188,000. Scatchard plots did not correlate with the antibody titres. The Ka values varied from 2.6 to 6.7 x 10(10) litres/mol. For maximum sensitivity the highest Ka (titre 1/66,000) was chosen. A tracer concentration of 22 pmol/l, an antiserum dilution of 1/100,000, and a sample volume of 0.1 ml were used. At an antiserum dilution of 1 in 100,000 the cyanocobalamin binding of the rabbit serum was diluted out. The assay showed excellent correlation with the microbiological assay, with 100% recovery of added vitamin B12. Levels of cross-reactivity for dicyanide cobinamide and hydroxocobalamin were 9.8 and 8.1%, respectively. CONCLUSIONS: The development of this immunoassay permits the measurement of serum vitamin B12 without important interference from cobalamin analogues, related corrinoids, and non-specific binders.

Animals↗
PubMed1997

Structure-based perspectives on B12-dependent enzymes.

Two X-ray structures of cobalamin (B12) bound to proteins have now been determined. These structures reveal that the B12 cofactor undergoes a major conformational change on binding to the apoenzymes of methionine synthase and methylmalonyl-coenzyme A mutase: The dimethylbenzimidazole ligand to the cobalt is displaced by a histidine residue from the protein. Two methyltransferases from archaebacteria that catalyze methylation of mercaptoethanesulfonate (coenzyme M) during methanogenesis have also been shown to contain histidine-ligated cobamides. In corrinoid iron-sulfur methyltransferases from acetogenic and methanogenic organisms, benzimidazole is dissociated from cobalt, but without replacement by histidine. Thus, dimethylbenzimidazole displacement appears to be an emerging theme in cobamide-containing methyltransferases. In methionine synthase, the best studied of the methyltransferases, the histidine ligand appears to be required for competent methyl transfer between methyl-tetrahydrofolate and homocysteine but dissociates for reductive reactivation of the inactive oxidized enzyme. Replacement of dimethylbenzimidazole by histidine may allow switching between the catalytic and activation cycles. The best-characterized B12-dependent mutases that catalyze carbon skeleton rearrangement, for which methylmalonyl-coenzyme A mutase is the prototype, also bind cobalamin cofactors with histidine as the cobalt ligand, although other cobalamin-dependent mutases do not appear to utilize histidine ligation. It is intriguing to find that mutases, which catalyze homolytic rather than heterolytic cleavage of the carbon-cobalt bond, can use this structural motif. In methylmalonylCoA mutase a significant feature, which may be important in facilitating homolytic cleavage, is the long cobalt-nitrogen bond linking histidine to the co-factor. The intermediate radical species generated in catalysis are sequestered in the relatively hydrophilic core of an alpha/beta barrel domain of the mutase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

Reddish Escherichia coli cells caused by overproduction of Bacillus stearothermophilus uroporphyrinogen III methylase: cloning, sequencing, and expression of the gene.

During shotgun cloning of an amylase gene, we found a transformant of Escherichia coli with a reddish color. The transformant produced highly water-soluble red pigments the molecular masses of which were less than 3000. The plasmid harbored by the transformant contained a DNA fragment derived from a strain of Bacillus stearothermophilus. Truncation of the insert DNA showed that an 1.1-kbp Sau 3A-SalI fragment was responsible for the reddish colony. An open reading frame was found in the nucleotide sequence of the 1.1-kbp DNA fragment. The production of the red pigment was accompanied by a colorless 28-kDa protein. The sequence of the 28-kDa protein was highly homologous to bacterial uroporphyrinogen III methylases participating in corrinoid biosynthesis. The 28-kDa protein was found to be a thermostable uroporphyrinogen III methylase.

Amino Acid Sequence↗
PubMedDate not supplied

On the reaction of methylmercuric hydroxide with methylbabalamin.

The methylmercury-induced dealkylation of the corrinoid coenzyme methylcobalamin, yielding aquocobalamin and dimethylmercury as products, was studied spectrophotometrically at 350 nm using water as a solvent. Rate data were determined for the pH 7-9 region and also at pH 3.37. Evidence is provided which shows that CH3Hg+ serves as the species which accepts the methyl group and also that Hg2+ is methylated more rapidly than CH3Hg+ is.

Chemical Phenomena↗
PubMedDate not supplied

Synthesis of 4-aza-5,6-dimethylbenzimidazole and biosynthetic preparation of 4- and 7-aza-5,6-dimethylbenzimidazolylcobamide.

We report on the preparation of 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide. These vitamin B12-analogs were required as reference compounds for comparison with a corrinoid previously isolated in small amounts for Eubacterium limosum grown in the presence of 4(5)-aminoimidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was synthesized from N-1-benzyl-4-nitroimidazole which was reduced to N-1-benzyl-4-aminoimidazole and condensed with 1-dimethylamino-2-methylbutan-3-one to yield N-1-benzyl-4-aza-5,6-dimethylbenzimidazole. The benzyl group of this compound was split off by catalytic hydrogenation to form 4(7)-aza-5,6-dimethylbenzimidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was transformed by a growing culture of Propionibacterium shermanii into 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide. Both vitamin B12-analogs were almost as active as Vitamin B12 in a growth test with the vitamin B12-dependent Escherichia coli-mutant DSM 4261.

Cobamides↗
PubMed2004

Occurrence of vitamin B12 in green, blue, red, and black tea leaves.

Vitamin B12 contents of green (0.046-0.263 and 0.125-0.535 microg/100 g dry weight), blue (0.068-0.081 and 0.525-0.528 microg/l00 g dry weight), red (0.061 and 0.663 microg/100 g dry weight), and black (0.104-0.859 and 0.305-1.20 microg/100 dry weight) tea leaves were obtained by intrinsic factor-chemiluminescence and microbiological methods, respectively. Although vitamin B12 was found in all tea leaves tested by both assay methods, the higher values by the microbiological method were not due to occurrence of both deoxyribosides and deoxynucleotides (known as an alkali-resistant factor), but may have been due to that of inactive corrinoid compounds for mammals in the tea leaves.

Camellia sinensis↗
PubMed2001

Purification, cloning, and sequencing of an enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from Clostridium bifermentans DPH-1.

An enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from cell-free extracts of Clostridium bifermentans DPH-1 was purified, cloned, and sequenced. The enzyme catalyzed the reductive dechlorination of PCE to cis-1,2-dichloroethylene via trichloroethylene, at a Vmax and Km of 73 nmol/mg protein and 12 microM, respectively. Maximal activity was recorded at 35 degrees C and pH 7.5. Enzymatic activity was independent of metal ions but was oxygen sensitive. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. The molecular mass of the native enzyme was estimated to be approximately 70 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) revealed molecular masses of approximately 35 kDa and 35.7 kDa, respectively. A broad spectrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropane, and 1,1,2-trichloroethane) was degraded. With degenerate primers designed from the N-terminal sequence (27 amino acid residues), a partial sequence (81 bp) of the encoding gene was amplified by polymerase chain reaction (PCR) and sequenced. Southern analysis of C. bifermentans genomic DNA using the PCR product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase.

Amino Acid Sequence↗
PubMed2002

Reductive biodegradation of 1,2-dichloroethane by methanogenic granular sludge: perspectives for in situ remediation.

Granular methanogenic sludge was able to dechlorinate 1,2-dichloroethane (1,2-DCA) to ethene in UASB reactors. Ethanol served as the sole carbon and energy source. The average dechlorination rate measured on the basis of ethene production varied between 1.7 and 2.1 micromol 1,2-DCA/(h.gVSS) (46.7 and 57.4 mg/L.d). In order to elucidate the microbial origin of this bioconversion, enrichment cultures of the methanogenic sludge were prepared with different carbon and electron sources: pyruvate, lactate, H2/CO2, ethanol and formate. Dithiothreitol (DTT) was the strong reductant in order to increase the negative redox potential in the media. A homo-acetogenic gram-positive strain could be isolated in the presence of formate. 16S rRNA of the isolated strain showed that the bacterium was closely related (99.7%) to Acetobacterium wieringae. The strain also grew on pyruvate, lactate, H2/CO2 and ethanol, although dechlorination rates of 1,2-DCA were at least 5 times higher when formate was the (only) electron source. Average conversion rates reached 3 micromol/(h.g(dry cells)) and appeared t relate to cometabolic biocatalysis on the corrinoid centers of the homo-acetogenic strain. Some perspectives of anaerobic in situ bioremediation of groundwater polluted with chloroethanes are presented.

Acetobacteraceae↗
PubMedDate not supplied

[The biology of aerobic methylobacteria capable of degrading halomethanes].

Recent data on the biology of aerobic methylotrophic bacteria capable of utilizing toxic halogenated methane derivatives as sources of carbon and energy are reviewed, with particular emphasis on the taxonomic, physiological, and biochemical diversity of mono- and dihalomethane-degrading methylobacteria and the enzymatic and genetic aspects of their primary metabolism. The initial steps of chloromethane dehalogenation to formate and HCl through a methylated corrinoid and methyletrahydrofolate are catalyzed by inducible cobalamin methyl transferase, made up of two proteins (CmuA and CmuB) encoded by the cmuA and cmuB genes. At the same time, the primary dehalogenation of dichloromethane to formaldehyde and HCl is catalyzed by cytosolic glutathione transferase with S-chloromethylglutathione as an intermediate. The latter enzyme is encoded by the structural dcmA gene and is under the negative control of the regulatory dcmR gene. In spite of considerable progress in the study of halomethane dehalogenation, some aspects concerning the structural and functional organization of this process and its regulation remain unknown, including the mechanisms of halomethane transport, the release of toxic dehalogenation products (S-chloromethylglutathione, CH2O, and HCl) from cells, and the maintenance of intracellular pH. Of particular interest is quantitative evaluation of the ecophysiological role of aerobic methylobacteria in the mineralization of halomethanes and protection of the biosphere from these toxic pollutants.

Formaldehyde↗
PubMedDate not supplied

[The aeration-dependent effect of vitamin B12 on DNA biosynthesis in Methylobacterium dichloromethanicum].

The effect of vitamin B12 (cobalamin) on DNA biosynthesis in Methylobacterium dichloromethanicum was studied. When cultivated in media with methanol or dichloromethane, the bacterium produced approximately 10 micrograms corrinoids per g dry biomass, compared to about 7 micrograms/g when cultivated on ethanol or succinate. Exogenous adenosylcobalamin (AdoCbl) stimulated DNA biosynthesis in M. dichloromethanicum cells grown under poor aeration, the effect being mediated by AdoCb1-linked ribonucleotide reductase. In vitro studies showed that M. dichloromethanicum also has AdoCbl-independent ribonucleotide reductase. Under good aeration, exogenous AdoCbl had no effect on DNA biosynthesis, while hydroxyurea suppressed it. These data suggest that AdoCbl-independent ribonucleotide reductase, which is likely to be activated by oxygen, plays an important part in DNA biosynthesis when M. dichloromethanicum is cultured with good aeration, whereas AdoCbl-dependent ribonucleotide reductase is active under the conditions of poor aeration.

Aerobiosis↗
PubMed1991

Biosynthesis of 5-hydroxybenzimidazolylcobamid (factor III) in Methanobacterium thermoautotrophicum.

Cultures of Methanobacterium thermoautotrophicum were supplemented with 13C-labeled acetate or pyruvate, and the labeling pattern of the corrinoid, factor III, was established by 13C NMR spectroscopy. Complete 13C signal assignments were obtained by two-dimensional NMR experiments. The labeling pattern of factor III was analyzed by comparison with those of amino acids and nucleosides. The corrin ring system is derived from eight molecules of glutamate. The aminopropanol moiety is derived in a hitherto unknown pathway from pyruvate by reductive amination. The heterocyclic ring of hydroxybenzimidazole shares the labeling pattern of the imidazole ring of purines. The remaining four carbon atoms of the carbocyclic ring show the labeling signature of a carbohydrate with two of the carbons introduced from acetate and two from C-1 of pyruvate. However, erythrose can be ruled out as the specific precursor on the basis of a detailed investigation of aromatic amino acids indicating that erythrose 4-phosphate is obtained by reductive carboxylation of a triose precursor and not by the pentose phosphate cycle.

Acetates↗
PubMed1989

Cobalamin analogues in plasma. An in vitro phenomenon?

In the course of investigations on the source of cobalamin analogues in human plasma six kits for measuring serum cobalamins were studies to determine if analogues can arise in vitro. High pressure liquid chromatography (HPLC) were used to estimate tracer stability during extraction procedures. Results showed that up to 60 percent of the radioactive cyanocobalamin was degraded. Alkaline pH increases the degradation; this effect is further intensified by addition of dithiothreitol. Implications for the cobalamin assay is discussed. Determination of cobalamin analogues by the difference of concentrations determined from assays using haptocorrin ("total corrinoids") and instrinsic factor ("true cobalamins") as binders may be misleading because the affinity of possible analogues for the assay binder is unknown. Thus quantification of the analogues could be erroneous. The need for definitive assay methods for cobalamin and its analogues is stressed.

Humans↗