The electrochemistry of vitamin B12

Lexa, Doris; Savéant, Jean Michel

doi:10.1021/ar00091a001

Cited by 386 publications

(364 citation statements)

References 38 publications

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“…N-terminal sequence analysis of the first 15 amino acids of the purified protein yielded the sequence TTLSCKVTSVEAITD, a perfect match to the E. coli Fre (encoded by the fre gene) (11,29). This finding was surprising because this enzyme was not expected to catalyze the reduction of cob(II)alamin to cob(I)alamin given the extremely low redox potential of the cob(II)alamin/cob(I)alamin couple (E 0 Ј ϭ Ϫ0.61V [1,19]). Noteworthy is the fact that this reduction was monitored using in vitro alkylation assays.…”

Section: Resultsmentioning

confidence: 99%

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

Fonseca

Escalante‐Semerena

2000

J Bacteriol

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Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His- The biologically active, coenzymic form of cobalamin (Cbl) contains a 5Ј-deoxyadenosyl (Ado) group as the upper axial ligand (Fig. 1). Formation of the C-Co bond between the cobalt ion and the upper ligand requires that the cobalt ion be reduced to its ϩ1 oxidation state. Reduction of Co(III) to Co(I) is thought to proceed in two consecutive one-electron reductions catalyzed by cob(III)alamin reductase (EC 1.6.99.8) and by cob(II)alamin reductase (EC 1.6.99.9) (36) (Fig. 2). The product of cob(II)alamin reductase, a Co(I) corrinoid, is the substrate for the ATP:co(I)rrinoid adenosyltransferase (CobA) (EC 2.5.1.17) enzyme that generates the C-Co bond. This bond is very labile and needs to be reformed to maintain activity. Hence, this branch of the Ado-Cbl biosynthetic pathway is essential for coenzyme recycling.In Salmonella enterica serovar Typhimurium LT2, the CobA enzyme responsible for the last step of the pathway has been isolated and partially characterized (30, 31). Although enzymic activities that can generate reduced corrinoids have been reported and in some cases isolated, the genes encoding the enzymes responsible for the reductive steps of the pathway have not been identified in any organism (2,15,36).Reduction of cob(III)alamin by crude cell-free extracts of Clostridium tetanomorphum and Propionibacterium freundenreichii showed an absolute requirement for added flavin cofactors (3,36). In these bacteria and in Pseudomonas denitrificans, NADH was the preferred source of electrons for the reaction (2). A system that included thiol compounds, a diaphorase enzyme, NADH, and flavin adenine dinucleotide (FAD) was used to reduce Cbl in P. freundenreichii (3,15). Interestingly, this complex-reducing system was replaced by FADH 2 when purified preparations of the adenosyltransferase from this organism were used. The cob(II)yrinic acid a,c-diamide reductase enzyme of P. denitrificans was isolated and shown to require the addition of flavin cofactors for activity (2). The purified enzyme was shown to reduce complete and incomplete Co(II) corrinoids. Here again, the gene encoding this activity was not identified.Extensive efforts to isolate mutants of S. enterica serovar Typhimurium LT2 defective in corrinoid reduction and adenosylation have failed to identify genetic loci other than cobA (9; M. V. Fonseca and J. C. Escalante-Semerena, unpublished results). Hence, we took a reverse genetics approach to the isolation of ...

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Section: Resultsmentioning

confidence: 99%

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

Fonseca

Escalante‐Semerena

2000

J Bacteriol

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“…Therefore, if there is no protection from oxygen, OH2/OHCbl would be readily formed on CblC during the journey to mitochondria. The reduction of OH2/OHCbl is not an issue because of the high redox potential of + 200 mV (21) and can even be directly used for enzyme cofactor synthesis (19).…”

Section: Discussionmentioning

confidence: 99%

Protection of aquo/hydroxocobalamin from reduced glutathione by a B₁₂ trafficking chaperone

Jeong

Ha²,

Kim³

2011

BMB Reports

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“…Very negative potentials (E ϭ approximately Ϫ1 V versus the standard hydrogen electrode) are needed for reducing methyl-and 5Ј-deoxyadenosylcobalamin (19,20), which are considered to be outside the range of biological reductants. Based on its enzymatic activity, we propose to rename the MMACHC protein cyanocobalamin decyanase.…”

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Decyanation of vitamin B ₁₂ by a trafficking chaperone

Kim

Gherasim

Banerjee

2008

Proc. Natl. Acad. Sci. U.S.A.

192

168

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The mystery of how the cyanide group in vitamin B12 or cyanocobalamin, discovered 60 years ago, is removed, has been solved by the demonstration that the trafficking chaperone, MMACHC, catalyzes a reductive decyanation reaction. Electrons transferred from NADPH via cytosolic flavoprotein oxidoreductases are used to cleave the cobalt-carbon bond with reductive elimination of the cyanide ligand. The product, cob(II)alamin, is a known substrate for assimilation into the active cofactor forms, methylcobalamin and 5 -deoxyadenosylcobalamin, and is bound in the ''base-off'' state that is needed by the two B12-dependent target enzymes, methionine synthase and methylmalonyl-CoA mutase. Defects in MMACHC represent the most common cause of inborn errors of B12 metabolism, and our results explain the observation that fibroblasts from these patients are poorly responsive to vitamin B12 but show some metabolic correction with aquocobalamin, a cofactor form lacking the cyanide ligand, which is mirrored by patients showing poorer clinical responsiveness to cyano-versus aquocobalamin.cobalamin ͉ flavin oxidoreductase ͉ methylmalonic aciduria ͉ homocystinuria ͉ cyanide V itamin B 12 (or cyanocobalamin, CNCbl) was discovered exactly 60 years ago in the laboratories of Folkers and Smith as the anti-pernicious anemia factor (1, 2), and its structure was revealed a few years later by the crystallographic studies in the Hodgkin laboratory (3). B 12 is one of the most complex cofactors in nature and belongs to the family of tetrapyrrolic-derived macrocyclic compounds. Five of the six ligands to the central cobalt atom in free B 12 are endogenous, being provided by the corin ring itself (Fig. 1A). In CNCbl, the sixth ligand, which occupies the upper axial position, is a cyano group. Although the biological origin of the cyano group in vitamin B 12 is unknown, it is the cofactor form commonly used in multivitamin formulations and one that individuals on vitamin supplementation are regularly exposed to. Decyanation of CNCbl is a prerequisite for its conversion to the active cofactor forms used by mammals in which the cyano ligand is replaced by a methyl group (in methylcobalamin) or a 5Ј-deoxyadenosyl group (in coenzyme B 12 or 5Ј-deoxyadenosylcobalamin) (Fig. 1B).Defects in B 12 metabolism are inherited as autosomal recessive disorders and are classified as belonging to one of eight genetic complementation groups, cblA-G and mut (4). Two loci, cblG and mut, encode B 12 -dependent enzymes: methionine synthase, which is cytoplasmic, and methylmalonyl-CoA mutase, which is mitochondrial. The remaining loci encode functions needed for intercompartmental trafficking and assimilation of the cofactor into its active forms. B 12 is present at relatively low concentrations in human tissues (5) and is reactive in all three of its biologically relevant oxidation states, factors that pose challenges for its unescorted delivery to target enzymes (6). The problem is further exacerbated by the use of the ''base-off/Hison'' conformation of the cofactor ...

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The electrochemistry of vitamin B12

Cited by 386 publications

References 38 publications

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

Protection of aquo/hydroxocobalamin from reduced glutathione by a B₁₂ trafficking chaperone

Decyanation of vitamin B ₁₂ by a trafficking chaperone

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The electrochemistry of vitamin B12

Cited by 386 publications

References 38 publications

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

Protection of aquo/hydroxocobalamin from reduced glutathione by a B12 trafficking chaperone

Decyanation of vitamin B 12 by a trafficking chaperone

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Protection of aquo/hydroxocobalamin from reduced glutathione by a B₁₂ trafficking chaperone

Decyanation of vitamin B ₁₂ by a trafficking chaperone