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PubMed1998

Tetrahydrofolate serves as a methyl acceptor in the demethylation of dimethylsulfoniopropionate in cell extracts of sulfate-reducing bacteria.

Tetrahydrofolate was shown to function as a methyl acceptor in the anaerobic demethylation of dimethylsulfoniopropionate to methylthiopropionate in cell extracts of the sulfate-reducing bacterium strain WN. Dimethylsulfoniopropionate-dependent activities were 0.56 micromol methyltetrahydrofolate min-1 (mg protein)-1 and were higher than required to explain the growth rate of strain WN on dimethylsulfoniopropionate. The reaction did not require ATP or reductive activation by titanium(III)-nitrilotriacetic acid. Preincubation of the extract under air significantly decreased the activity (35% loss in 3 h). Three other dimethylsulfoniopropionate-demethylating sulfate reducers, Desulfobacterium niacini, Desulfobacterium vacuolatum, and Desulfobacterium strain PM4, had dimethylsulfoniopropionate:tetrahydrofolate methyltransferase activities of 0.16, 0.05, and 0.24 micromol min-1 (mg protein)-1, respectively. No methyltransferase activity to tetrahydrofolate was found with betaine as a substrate, not even in extracts of betaine-grown cells of these sulfate reducers. Dimethylsulfoniopropionate demethylation in cell extracts of strain WN was completely inhibited by 0.5 mM propyl iodide; in the light, the inhibition was far less strong, indicating involvement of a corrinoid-dependent methyltransferase.

Biodegradation, Environmental↗
PubMed1998

Purification and characterization of the tetrachloroethene reductive dehalogenase of strain PCE-S.

The membrane-associated tetrachloroethene reductive dehalogenase from the tetrachloroethene-reducing anaerobe, strain PCE-S, was purified 165-fold to apparent homogeneity in the presence of the detergent Triton X-100. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor, showing a specific activity of 650 nkat/mg protein. The apparent Km values of the enzyme for tetrachloroethene, trichloroethene, and methyl viologen were 10 microM, 4 microM, and 0.3 mM, respectively. SDS-PAGE revealed a single protein band with an apparent molecular mass of 65 kDa. The apparent molecular mass of the native enzyme was 200 kDa as determined by gel filtration. Tetrachloroethene dehalogenase contained 0.7 +/- 0.3 mol corrinoid, 1.0 +/- 0.3 mol cobalt, 7.8 +/- 0.5 mol iron, and 10.3 +/- 2.0 mol acid-labile sulfur per mol subunit. The pH optimum was approximately 7.2, and the temperature optimum was approximately 50 degrees C. The dehalogenase was oxygen-sensitive with a half-life of approximately 50 min. The N-terminal amino acid sequence of the enzyme was determined, and no significant similarity was found to any part of the amino acid sequence of the tetrachloroethene (PCE) reductive dehalogenase from Dehalospirillum multivorans.

Bacteria, Anaerobic↗
PubMed1998

Tetramethylammonium:coenzyme M methyltransferase system from methanococcoides sp

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens.

Journal Article↗
PubMed2004

Autocatalytic activation of acetyl-CoA synthase.

Acetyl-CoA synthase (ACS identical with ACS/CODH identical with CODH/ACS) from Moorella thermoacetica catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group of a corrinoid-iron-sulfur protein (CoFeSP). A time lag prior to the onset of acetyl-CoA production, varying from 4 to 20 min, was observed in assay solutions lacking the low-potential electron-transfer agent methyl viologen (MV). No lag was observed when MV was included in the assay. The length of the lag depended on the concentrations of CO and ACS, with shorter lags found for higher [ACS] and sub-saturating [CO]. Lag length also depended on CoFeSP. Rate profiles of acetyl-CoA synthesis, including the lag phase, were numerically simulated assuming an autocatalytic mechanism. A similar reaction profile was monitored by UV-vis spectrophotometry, allowing the redox status of the CoFeSP to be evaluated during this process. At early stages in the lag phase, Co(2+)FeSP reduced to Co(+)FeSP, and this was rapidly methylated to afford CH(3)-Co(3+)FeSP. During steady-state synthesis of acetyl-CoA, CoFeSP was predominately in the CH(3)-Co(3+)FeSP state. As the synthesis rate declined and eventually ceased, the Co(+)FeSP state predominated. Three activation reductive reactions may be involved, including reduction of the A- and C-clusters within ACS and the reduction of the cobamide of CoFeSP. The B-, C-, and D-clusters in the beta subunit appear to be electronically isolated from the A-cluster in the connected alpha subunit, consistent with the ~70 A distance separating these clusters, suggesting the need for an in vivo reductant that activates ACS and/or CoFeSP.

Acetate-CoA Ligase↗
PubMed2004

Acetyl-coenzyme A synthase: the case for a Ni(p)(0)-based mechanism of catalysis.

Acetyl-CoA synthase (also known as carbon monoxide dehydrogenase) is a bifunctional Ni-Fe-S-containing enzyme that catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-coenzyme A from CO, CoA, and a methyl group donated by a corrinoid iron-sulfur protein. The active site for the latter reaction, called the A-cluster, consists of an Fe(4)S(4) cubane bridged to the proximal Ni site (Ni(p)), which is bridged in turn to the so-called distal Ni site. In this review, evidence is presented that Ni(p) achieves a zero-valent state at low potentials and during catalysis. Ni(p) appears to be the metal to which CO and methyl groups bind and then react to form an acetyl-Ni(p) intermediate. Methyl group binding requires reductive activation, where two electrons reduce some site on the A-cluster. The coordination environment of the distal Ni suggests that it could not be stabilized in redox states lower than 2+. The rate at which the [Fe(4)S(4)](2+) cubane is reduced is far slower than that at which reductive activation occurs, suggesting that the cubane is not the site of reduction. An intriguing possibility is that Ni(p)(2+) might be reduced to the zero-valent state. Reinforcing this idea are Ni-organometallic complexes in which the Ni exhibits analogous reactivity properties when reduced to the zero-valent state. A zero-valent Ni stabilized exclusively with biological ligands would be remarkable and unprecedented in biology.

Acetate-CoA Ligase↗
PubMed1984

Significance of isomerization in hydroxocobalamin.

Hydroxocobalamin is present in fairly large proportions both in foods and in the human body and apparently plays an important biological role. Since cyanocobalamin seems to play hardly any significant biochemical role in healthy humans, several physicians prefer to administer hydroxocobalamin to vitamin B12 deficient patients. We find that hydroxocobalamin in solution isomerizes very readily at room and lower temperatures. Our observations raise the question whether "Mother Nature" has gone awry in using an easily convertible substance like hydroxocobalamin or that the new isomeric forms play some significant role. These observations may also have a bearing on the reported occurrence of unidentified corrinoids in animal tissues, human red cells, liver and brain.

Chemical Phenomena↗
PubMed1991

Coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Protection by the substrate from inactivation by light.

Partially purified 2-methyleneglutarate mutase from Clostridium barkeri was separated from 3-methylitaconate delta-isomerase by treatment with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) followed by FPLC on the anion exchange column Mono Q in the presence of the detergent. When purified in the dark, the active mutase contained a corrinoid, most probably coenzyme B12. The enzyme was inactivated by light at the same wavelength (lambda less than 620 nm) and rate as free coenzyme B12. The rate was not influenced by oxygen or by temperature (0-37 degrees C). Reactivation of up to 50% of the original activity was achieved by incubation with coenzyme B12 and dithiothreitol. The substrates 2-methyleneglutarate (up to 40 mM) or (R)-3-methylitaconate specifically protected the enzyme from inactivation by visible light. This effect was enhanced 3-fold by raising the temperature from 0 degrees C to 37 degrees C. The data indicate that during catalysis, the Co-C bond of the coenzyme is cleaved and cannot be affected any more by light.

Carbon-Carbon Double Bond Isomerases↗
PubMed1998

Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense.

The membrane-bound 3-chloro-4-hydroxyphenylacetate (Cl-OHPA) reductive dehalogenase from the chlorophenol-reducing anaerobe Desulfitobacterium hafniense was purified 11.3-fold to apparent homogeneity in the presence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive dechlorination of Cl-OHPA to 4-hydroxyphenylacetate with reduced methyl viologen as the electron donor at a specific activity of 103.2 nkat/mg protein. SDS-PAGE revealed a single protein band with an apparent molecular mass of 46.5 kDa. The enzyme contained 0.68 +/- 0.2 mol corrinoid, 12.0 +/- 0.7 mol iron, and 13.0 +/- 0.7 mol acid-labile sulfur per mol subunit. The N-terminal amino acid sequence of the enzyme was determined and no significant similarity was found to any protein present in the gene bank.

Acetates↗
PubMed1999

The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH.

In methanogenic archaea the transfer of the methyl group of N5-methyltetrahydromethanopterin to coenzyme M is coupled with energy conservation. The reaction is catalyzed by a membrane associated multienzyme complex composed of eight different subunits MtrA-H. The 23 kDa subunit MtrA harbors a corrinoid prosthetic group which is methylated and demethylated in the catalytic cycle. We report here that the 34 kDa subunit MtrH catalyzes the methylation reaction. MtrH was purified and shown to exhibit methyltetrahydromethanopterin:cob(I)alamin methyltransferase activity. Sequence comparison revealed similarity of MtrH with MetH from Escherichia coli and AcsE from Clostridium thermoaceticum: both enzymes exhibit methyltetrahydrofolate:cob(I)alamin methyltransferase activity.

Amino Acid Sequence↗
PubMed2000

Halorespiring bacteria-molecular characterization and detection.

Recently, a rapidly increasing number of bacteria has been isolated that is able to couple the reductive dehalogenation of various halogenated aromatic and aliphatic compounds like chlorophenols and tetrachloroethene to energy conservation by electron-transport-coupled phosphorylation. The potential of these halorespiring bacteria for innovative clean-up strategies of polluted anoxic environments has greatly stimulated efforts to unravel the molecular basis of the novel respiratory chains they possess. The thorough characterization of halorespiratory key components at the physiological, biochemical and molecular genetic level has revealed both structural and functional similarity of chloroaryl- and chloroalkyl-respiratory chains from different phylogenetically distinct microorganisms. The reductive dehalogenases from halorespiring bacteria were found to comprise a novel class of corrinoid-containing Fe/S-proteins. Sensitive molecular methods for monitoring both presence and fate of halorespiring bacteria have been developed, which will be instrumental for the design and maintenance of optimised in situ bioremediation processes.

Journal Article↗
PubMed1979

Cryptofluorescent analogs of cobalamin coenzymes: synthesis and characterization.

Upper-axial (beta-position) ligand analogs of the B12 coenzymes 5'-deoxy-5'-adenosylcob(III)alamin and methylcob(III)alamin have been synthesized by reaction of the 5'-chloro-5'-deoxy derivatives of fluorescent nucleosides (1,N6-ethenoadenosine, formycin, 2-amino-nebularine, and 2,6-diaminonebularine) and a fluorescent alkyl halide (dansylamidopropyl chloride) with cob(I)alamin. These analogs were nonfluorescent, but fluorescent products could be generated by photolysis or cyanolysis of the carbon-cobalt bonds. Under anaerobic and aerobic conditions, the major fluorescent photolysis products of 1,N6-ethenoadenosylcob(III)alamin were 1,N6-etheno-5',8-cyclic-5'-deoxyadenosine, and the 5'-aldehyde of 1,N6-ethenoadenosine, respectively. The cryptofluorescent property of these analogs was utilized to follow the kinetics of aerobic photolysis. First-order rate constants determined by this method were comparable to those obtained spectrophotometrically [via appearance of of aquacob(III)alamin]. Pseudo-first-order rate constants determined fluorometrically for the cyanolysis (at 25 degrees C) of 1,N6-ethenoadenosylcob(III)alamin, 2,6-diaminonebularinylcob(III)alamin, 2-aminonebularinylcob(III)alamin and formycinylcob(III)alamin were 5.8 X 10(-2), 2 X 10(-2), 1.8 X 10(-2), and 3 X 10(-5) min-1, respectively; values in good agreement were obtained spectrophotometrically (via appearance of dicyanocobalamin). Dansylamidopropylcob(III)alamin was stable in the presence of cyanide. The nucleoside alpha-ribazole is fluorescent in the free state but nonfluorescent when present as the lower axial (alpha-position) ligand in cobalamin coenzymes. Thus fluorescence of ligands in both the alpha- and beta-positions of cobalamins is quenched, probably as a result of intramolecular energy transfer between the ligands and the nonfluorescent corrinoid.

Aerobiosis↗
PubMed2002

Synthesis of adenosylcobinamide 2-chlorophenyl phosphate, a zwitterionic cobinamide phosphate analog of adenosylcobalamin en route to crystallizable cobinamides.

The design and implementation of a new, higher yield synthetic method for synthesizing zwitterionic cobinamide phosphates is described. Adenosylcobinamide 2-chlorophenyl phosphate, beta-AdoCbi-PAr -- a 5,6-dimethylbenzimidazole-free adenosylcobalamin analog, where a 2-chlorophenyl group replaces the ribofuranose and 5,6-dimethylbenzimidazole moieties -- is prepared in tens of milligram quantities, quantities sufficient for crystallization and enzyme trials, amounts 100-fold greater than previously available. The use of (31)P NMR spectroscopy to follow reactions directly, the use of control reactions to learn how to reduce reactant water content, and the use of reaction solvents that completely dissolved the corrinoid reactants were crucial for developing this new synthetic route. beta-AdoCbi-PAr was synthesized in 10% overall isolated yield from cyanocobinamide. Cyanocobinamide was converted to cyanocobinamide 2-chlorophenyl phosphate by direct phosphorylation with 2-chlorophenyl phosphodi-(1,2,4-triazolide) in 25% isolated yield and > or = 98% purity. Sodium borohydride reduction of cyanocobinamide 2-chlorophenyl phosphate and reaction with 5'-chloro-5'-deoxy-adenosine produced beta-AdoCbi-PAr in 42% yield and > or = 98% purity. These compounds were characterized by HPLC, (1)H and (31)P NMR, UV-visible spectroscopy, and liquid secondary ionization mass spectroscopy.

Amides↗
PubMed2001

A gene, cobA + hemD, from Selenomonas ruminantium encodes a bifunctional enzyme involved in the synthesis of vitamin B12.

Coenzymes derived from vitamin B12 (cyanocobalamin) are particularly important for core metabolism in ruminant animals. Selenomonas ruminantium, a Gram-positive obligate anaerobe isolated from cattle, is the main contributor of vitamin B12 to such ruminant animals. In nature, there are both aerobic and anaerobic pathways for B12 synthesis - the latter is only partly elucidated. Until now, there has been no investigation of B12 synthesis in S. ruminantium, which must use an anaerobic pathway. This paper reports the cloning of the chromosomal operon from S. ruminantium which is responsible for the first committed steps in corrinoid synthesis. Five open reading frames were found in the cloned fragment. All deduced amino acid sequences had similarity to defined proteins in the databases that are involved in porphyrin and corrin synthesis. Of particular interest is the gene designated cobA + hemD, which encodes a single polypeptide possessing two catalytic functions - uroporphyrinogen III synthase and uroporphyrinogen III 2,7-methyltransferase. This enzyme converts hydroxymethylbilane to precorrin-2. The functions of the protein coded by cobA + hemD were established by heterologous expression in Escherichia coli. The CobA activity has been demonstrated for three distinct types of proteins - monofunctional, bifunctional with siroheme formation and, this report, bifunctional with uroporphyrinogen III synthesis. The type found in S. ruminantium (cobA + hemD) is probably restricted to obligately anaerobic fermentative bacteria.

Alkyl and Aryl Transferases↗
PubMed2002

Anaerobic benzene biodegradation--a new era.

Benzene is biodegraded in the absence of oxygen under a variety of terminal electron-accepting conditions. However, the mechanism by which anaerobic benzene degradation occurs is unclear. Phenol and benzoate have been consistently detected as intermediates of anaerobic benzene degradation, suggesting that the hydroxylation of benzene to phenol is one of the initial steps in anaerobic benzene degradation. The conversion of phenol to benzoate could then occur by the carboxylation of phenol to form 4-hydroxybenzoate followed by the reductive removal of the hydroxyl group to form benzoate. 13C-Labeling studies suggest that the carboxyl carbon of benzoate is derived from one of the carbons of benzene. Although the fumarate addition reaction is commonly used to activate many hydrocarbons for anaerobic degradation, the large activation energy required to remove hydrogen from the benzene ring argues against such an approach for anaerobic benzene metabolism. The alkylation of benzene to toluene has been detected in several mammalian tissues, and offers an interesting alternate hypothesis for anaerobic benzene degradation in microbial systems. In support of this, anaerobic benzene degradation by Dechloromonas strain RCB, the only known species to degrade benzene in the absence of oxygen, is stimulated by the addition of vitamin B12 and inhibited by the addition of propyl iodide which is consistent with the involvement of a corrinoid enzymatic step. Alkylation of benzene to toluene is also consistent with labeling data that suggests that the carboxyl carbon of benzoate is derived from one of the benzene carbons. However, it is difficult to envision how phenol would be formed if benzene is alkylated to toluene. As such, it is possible that diverse mechanisms for anaerobic benzene degradation may be operative in different anaerobic microorganisms.

Anaerobiosis↗
PubMed2001

Microbial dehalogenation.

Novel dehalogenases have been identified recently in various bacteria that utilise halogenated substrates. X-ray studies and sequence analysis have revealed insight into the molecular mechanisms of hydrolytic dehalogenases. Furthermore, genetic and biochemical studies have indicated that reductive dehalogenases are extra-cytoplasmic corrinoid-containing iron-sulphur proteins. Sequence analysis and mutagenesis studies indicate that several dehalogenases are homologous to enzymes that carry out transformations on non-halogenated substrates.

Aerobiosis↗
PubMed2004

Fe(III) and Co(III) centers with carboxamido nitrogen and modified sulfur coordination: lessons learned from nitrile hydratase.

Nitrile hydratase (NHase) is a non-heme Fe(III) or non-corrinoid Co(III) metalloenzyme with an unprecedented coordination sphere comprising deprotonated carboxamido nitrogens and modified Cys-S (-SO(-) and -SO(2)(-)) sulfurs. We have synthesized model complexes derived from designed ligands that contain these donor groups. The model complexes mimic almost all the intrinsic properties of the unique M(III) (M = Fe, Co) active site of NHase. Even a functional Co(III) model has been synthesized that hydrolyzes nitriles catalytically at pH close to the optimum pH of the enzyme. Our studies have provided insight into how the unusual donor atoms dictate the overall properties of the biological M(III) sites.

Amides↗
PubMed1984

Influence of metal substitution on vitamin B12 binding to human intrinsic factor and transcobalamins I and II.

Metal-free, zinc, copper, and rhodium analogues of vitamin B12 were synthesized to further characterize structural requirements for the binding to human intrinsic factor, transcobalamin I, and transcobalamin II. Binding affinities of the various analogues were studied by competition against cyano[57Co]cobalamin. When albumin-coated charcoal was used for the separation of free and bound corrinoids, the relative 50% inhibition indexes were determined. The influence of metal substitution was similar among the three binding proteins. For analogues with a strong coordinative linkage between the heterocyclic base and the central metal ion, similar to that with cobalt (e.g., zincobalamin and cyanorhodibalamin), the indexes range from 0.65 to 2.35 for all three binding proteins. Analogues in which coordination is impossible (hydrogenobalamin and dicyanorhodibalamin) exhibit markedly reduced binding with indexes between 10 and 160. Cupribalamin shows 50% inhibition indexes ranging from 2.3 to 5.0, thus suggesting a weak coordinative bond between the copper ion and the 5,6-dimethylbenzimidazole moiety. These results emphasize the importance of the coordinative linkage between the central metal ion and the nucleotide moiety for optimal recognition by vitamin B12 binding proteins.

Binding Sites↗
PubMed1985

Methyl transfer from methylcobalamin to thiols. A reinvestigation.

The methyl transfer from methylcobalamin to thiols has been reinvestigated. By use of methylcobalamin selectively enriched with 13C in the methyl moiety, the methyl transfer to thiols was followed by 13C NMR. The methyl transfer occurs in aqueous mildly alkaline (pH 8-12) solution, even in the complete absence of oxygen. 31P NMR and EPR studies demonstrate that cob(II)alamin is the final corrinoid product. However, the pH dependence of the methyl-transfer reaction from methylcobalamin to beta-mercaptoethanol is consistent only with a nucleophilic displacement of the methyl group by a thiolate anion, resulting in the heterolytic cleavage of the carbon-cobalt bond. Difference visible spectroscopic measurements of the reaction mixture suggest that cob(I)alamin is formed as an intermediate.

Anaerobiosis↗