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     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

Survival of Lactobacillus leichmannii in Relation to Vitamin B12 Assays

Since washed cells of Lactobacillus leichmannii ATCC 7830 frequently showed erratic results in vitamin B₁₂ assays, a depletion technique was used to stabilize the inoculum. The method consisted of incubating the twice-washed cells at 37 C for 45 min in distilled water. Survival and growth studies indicated that the stabilizing procedure (i) did not affect cell numbers, (ii) was optimal for depleting possible carry-over of vitamin B₁₂ or other nutrilites and reserves, and (iii) brought about a phasing of the cells of the inoculum which underwent logarithmic growth immediately on inoculation. This minimal period (45 min) was the same when the cells were incubated in dextrose-water. Survival of the cells in physiological saline was greater than in distilled water, but a longer time was required for stabilizing the inoculum in saline, which precluded its use in routine work. The prepared inoculum showed an improvement over the washed inoculum in that readings between duplicate and triplicate tubes agreed closely and the standard curves were reproducible whether acidimetric or turbidimetric methods were followed. No erratic results were noted in more than 1 year of testing.     

Towards the synthesis of light-stable coenzyme B12 analogs

The organometallic complex coenzyme B₁₂ (adenosyl cobalamin, AdoCbl) is not only an essential coenzyme in many biochemical reactions of most if not all living organisms but has lately been shown to play a crucial role in the regulation of B₁₂ related genes. As a consequence, coenzyme B₁₂ has been a target of intense research. However, the investigations of AdoCbl have often been hampered due to its high light-sensitivity leading to decomposition of the compound within a few seconds. Here, we describe a strategy to synthesize more light-stable coenzyme B₁₂ analogs, which show similar steric properties as adenosyl cobalamin. The synthesis, structural characterization as well as the pH dependent “base-on/base-off” behavior of cyanide bridged vitamin B₁₂ conjugates with either a cis-[(NH₃)₂Pt]²⁺ or an [enPt]²⁺ moiety, leading to cis-[(NH₃)₂PtCl-vitB₁₂]⁺ (1) and [enPtCl-vitB₁₂]⁺ (2) are reported. The subsequent reaction of cis-[(NH₃)₂PtCl-vitB₁₂]⁺ with the model nucleobase 9-methyladenine leads to the corresponding adduct, where the adenine moiety is coordinated to the Pt²⁺ center either via N1 or N7. This compound is light-stable and harbors the adenine moiety in the same distance of 5 Å above the corrin plane as present in the highly light-sensitive adenosyl cobalamin.     

Decreased Brain Levels of Vitamin B12 in Aging,Autism and Schizophrenia

Many studies indicate a crucial role for the vitamin B₁₂ and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B₁₂ status in the brain across the lifespan has not been previously investigated. Vitamin B₁₂ (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B₁₂ status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.     

Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions

Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5′-deoxyadenosylcobalamin (AdoCbl) or coenzyme B₁₂. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B₁₂ metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase K_M(ATP). ³¹P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the k_cat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.     

Isolation of a novel Pseudomonas species SP2 producing vitamin B12 under aerobic condition

Vitamin B₁₂ is a complex biomolecule that acts as a cofactor for a variety of enzymes in microbial metabolism. Pseudomonas denitrificans is exclusively used as an industrial strain for the production of vitamin B₁₂ under aerobic conditions. However, only a few strains of Pseudomonas have been reported to possess the capability of producing this vitamin and they are strongly patent-protected. To improve the applicability of the vitamin B₁₂-producing microorganisms, a new isolate was obtained from municipal waste samples and characterized for its biological properties. The new isolate, designated as SP2, was identified to be a Pseudomonas species based on the sequence homology of its 16S rDNA. Pseudomonas species SP2 had essential genes for vitamin B₁₂ synthesis such as cobB and cobQ and produced a similar amount of vitamin B₁₂ (10.6 ± 0.05 μg/mL) as P. denitrificans ATCC 13867 in 24 h flask culture. SP2 grew well under aerobic condition with the maximum specific growth rate (µ _max ) of 0.91 ± 0.03/h, but showed a poor growth under micro-aerobic conditions. SP2 was resistant to antibiotics like streptomycin, carbenicillin, ampicillin, cefpodoxime, colistin, nalidixic acid and sparfloxacin. The ability of SP2 to grow faster and produce vitamin B₁₂ under aerobic conditions makes it a promising host for the production of some biochemicals requiring a coenzyme B₁₂-dependent enzyme, such as glycerol dehydratase.     

Multiple roles of ATP:cob(I)alamin adenosyltransferases in the conversion of B12 to coenzyme B12

Our mechanistic understanding of the conversion of vitamin B₁₂ into coenzyme B₁₂ (a.k.a. adenosylcobalamin, AdoCbl) has been substantially advanced in recent years. Insights into the multiple roles played by ATP:cob(I)alamin adenosyltransferase (ACA) enzymes have emerged through the crystallographic, spectroscopic, biochemical, and mutational analyses of wild-type and variant proteins. ACA enzymes circumvent the thermodynamic barrier posed by the very low redox potential associated with the reduction of cob(II)alamin to cob(I)alamin by generating a unique four-coordinate cob(II)alamin intermediate that is readily converted to cob(I)alamin by physiological reductants. ACA enzymes not only synthesize AdoCbl but also they deliver it to the enzymes that use it, and in some cases, enzymes in which its function is needed to maintain the fidelity of the AdoCbl delivery process have been identified. Advances in our understanding of ACA enzyme function have provided valuable insights into the role of specific residues, and into why substitutions of these residues have profound negative effects on human health. From an applied science standpoint, a better understanding of the adenosylation reaction may lead to more efficient ways of synthesizing AdoCbl.     

Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Effect of methionine and vitamin B-12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12

The effects of supplementation of growth medium with high concentrations of methionine (5 mm) and/or vitamin B₁₂ (10 nm) on the activities of five enzymes of the methionine regulon were measured in wild-type Escherichia coli K12, a metJ prototrophic and three metJ methionine auxotrophic derivatives. Growth on vitamin B₁₂ causes lowering of the activities of the non-B₁₂ methyltransferase while growth on methionine causes elevation of its activity in all four metJ mutants. The previous observation that this enzyme is not repressed by vitamin B₁₂ addition in metH mutants together with our observation that vitamin B₁₂ causes repression in mutants (metF) unable to synthesize the donor for homocysteine methylation supports the model of Kung et al. (10) that the holo-B₁₂-methyltransferase functions as a repressor of synthesis of the non-B₁₂-methyltransferase. Growth on methionine causes lowering of cystathionase activity, and growth on vitamin B₁₂ results in elevation of cystathionase activity in a metJ prototroph and one metJ auxotroph. The metJmetA strain (RG326) has a higher than normal level of cystathionase while the metJmetF strain (RG191) has lower than normal cystathionase activity. These results indicate the existence of a metJ independent system that modulates the activity of cystathionase possibly in response to changes in concentration of unidentified metabolite(s).     

The distribution of cobalt inT. subterraneum

Summary The distribution of cobalt in plants ofT. subterraneum grown on different levels of supply of cobalt and nitrate nitrogen has been measured, and the nodules and to a lesser extent the roots have been found to accumulate much more than the leaves.The distribution of vitamin-B₁₂-type compounds and of Co⁶⁰ in the nodule have also been determined following fractionation of the nodule components by hgh speed centrifugation. Most of the cobalt whether present as vitamin B₁₂, or as indicated by the measurement of Co⁶⁰, is present in the supernatant and in the bacteroids. The distribution of Co⁶⁰ is much the same in the nodules from both effective and non-effective strains. The quantities of vitamin B₁₂ present in both bacteroids and the supernatant are greatly increased, in relation to the fresh weight of the nodule tissue, when the supply of cobalt in the nutrient is raised.When Co⁶⁰ is incorporated into the plant a proportion of the activity is found to be present as vitamin B₁₂, free from bacteroids, within four days of addition. After this time the supernatant still contained about 90 per cent of the activity that entered. Of this some 40 per cent was present as ionic cobalt, 19 per cent as vitamin B₁₂ like compounds, and the remainder as a compound, presently termed Factor N, which is so far unidentified.Most of the data on which this paper is based are from the thesis submitted by S.B.W. for the degree of Ph. D. of the University of Nottingham.     

Effect of Yeast Extract and Vitamin B12 on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B₁₂, biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B₁₂ alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

Vitamin analysis of five planktonic microalgae and one macroalga

Vitamin analysis was carried out on five microalgae used in aquaculture:Tetraselmis suecica, Isochrysis galbana, Pavlova lutheri, Skeletonema costatum andChaetoceros calcitrans and one macroalga,Sargassum muticum, which is invasive on the Atlantic shores of France. Both liposoluble (provitamin A, E, K) and hydrosoluble (B₁, B₂, B₆, B₁₂, C, PP) vitamins were quantified. For most of them, greater amounts were obtained in the algal products than in the usual sources. On a dry weight basis,Tetraselmis suecica contained 4280 μg g^?1 provitamin A and 6323 μg g^?1 vitamin E,Pavlova lutheri 1162 μg g^?1 vitamin B₁₂ and 837 μg g^?1 vitamin C,Isochrysis galbana 2690 μg g^?1 vitamin PP and 183 μg g^?1 vitamin B₆, andSkeletonema costatum 710 μg g^?1 vitamin B₁.     

Mg2+-induced conformational changes in the btuB riboswitch from E. coli

Mg²⁺ has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B₁₂ (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B₁₂ transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg²⁺ and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg²⁺. With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg²⁺ (or low Mg²⁺ concentration) and the other appearing above ∼0.5 mM Mg²⁺. Distinct regions of the riboswitch exhibit different dissociation constants toward Mg²⁺, indicating a stepwise folding of the btuB RNA. Increasing the Mg²⁺ concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg²⁺ defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B₁₂, and switch the RNA conformation. Moreover, raising the Mg²⁺ concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg²⁺ indicates a crucial role played by Mg²⁺ for defining an active conformation of the riboswitch.     

Bile and detergent interaction with the radioassay for vitamin B12 binders using protein and dextran-covered charcoal

Bile acids and detergents such as Triton X-100, sodium dodecyl sulfate, and sodium deoxycholate interfered with the radioassay for vitamin B₁₂ binders. The effect was dependent on the concentration of the detergent. The detergents and bile acids were found, by use of [¹⁴C]glycocholic acid, to bind firmly to the covered charcoal in the assay, thereby blocking the binding of free vitamin B₁₂ to the charcoal. An increased ⁵⁷Co-labeled vitamin B₁₂ concentration was therefore observed in the final supernatant in the assay, giving rise to an apparent but false vitamin B₁₂-binding capacity.     

Megaloblastic Anemia Associated with Surgically Produced Gastrointestinal Abnormalities

Two of the mechanisms for vitamin B₁₂ deficiency, leading to megaloblastic anemia, are the result of surgically produced abnormalities of the gastrointestinal tract. The basic mechanism is different for each lesion.Total gastrectomy results in complete lack of intrinsic factor which is necessary for vitamin B₁₂ absorption. It is believed that if patients survive long enough and are not given prophylactic vitamin B₁₂ therapy, all would develop megaloblastic anemia.Intestinal anastomosis leading to stasis of intestinal contents, with overgrowth of bacteria may cause vitamin B₁₂ deficiency through bacterial interference with the utilization of vitamin B₁₂.Use of radioactive vitamin B₁₂ (cobalt⁶⁰-labeled B₁₂) has led to a better understanding of the pathogenesis of both types of megaloblastic anemia. The radioactive vitamin provides a useful tool for study of its absorption from the gastrointestinal tract.     

Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k _cat and k _cat/K _m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K _i values for d-glucose (83.3–357 mM) and d-xylose (15.6–70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K _t ^0.5?～?45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k _cat and k _cat/K _m on natural substrates xylobiose (407 s^?1, 138 s^?1?mM^?1), xylotriose (235 s^?1, 80.8 s^?1?mM^?1), and xylotetraose (146 s^?1, 32.6 s^?1?mM^?1).     

Vitamin B12-induced alterations in activities of α-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch)

Different doses of vitamin B₁₂ (0.25, 0.5, 1, 2 and 4 μg/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-α-glycerophosphate dehydrogenase (α-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The α-GPD activity increased at doses of 0.5, 1, 2 and 4 μg/g vitamin B₁₂. A dose of 0.5 μg/g vitamin B₁₂ induced less activity than higher doses. ME activity increased with 1, 2 and 4 μg/g of vitamin B₁₂/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B₁₂ administration. Cycloheximide treatment inhibited the vitamin B₁₂-induced increase in α-GPD and ME activity. Thus, vitamin B₁₂ is involved in the induction of some enzymes in fish brain.     

Regiospecificity of Chlorophenol Reductive Dechlorination by Vitamin B12s

Vitamin B₁₂, reduced by titanium (III) citrate to vitamin B_12s, catalyzes the reductive dechlorination of chlorophenols. Reductive dechlorination of pentachlorophenol and of all tetrachlorophenol and trichlorophenol isomers was observed. Reaction of various chlorophenols with vitamin B₁₂ favored reductive dechlorination at positions adjacent to another chlorinated carbon, but chlorines ortho to the hydroxyl group of a phenol were particularly resistant to reductive dechlorination, even if they were also ortho to a chlorine. This resulted in a reductive dechlorination pattern favoring removal of para and meta chlorines, which differs substantially from the pattern exhibited by anaerobic microbial consortia.