Biochemistry of B12-cofactors in human metabolism.

Vitamin B12, the "antipernicious anaemia factor", is a crystallisable cobalt-complex, which belongs to a group of unique "complete" corrinoids, named cobalamins (Cbl). In humans, instead of the "vitamin", two organometallic B12-forms are coenzymes in two metabolically important enzymes: Methyl-cobalamin, the cofactor of methionine synthase, and coenzyme B12 (adenosyl-cobalamin), the cofactor of methylmalonyl-CoA mutase. The cytoplasmatic methionine synthase catalyzes the transfer of a methyl group from N-methyl-tetrahydrofolate to homocysteine to yield methionine and to liberate tetrahydrofolate. In the mitochondrial methylmalonyl-CoA mutase a radical process transforms methylmalonyl-CoA (a remains e.g. from uneven numbered fatty acids) into succinyl-CoA, for further metabolic use. In addition, in the human mitochondria an adenosyl-transferase incorporates the organometallic group of coenzyme B12. In all these enzymes, the bound B12-derivatives engage (or are formed) in exceptional organometallic enzymatic reactions. This chapter recapitulates the physiological chemistry of vitamin B12, relevant in the context of the metabolic transformation of B12-derivatives into the relevant coenzyme forms and their use in B12-dependent enzymes.

Nitrous oxide-induced B12 deficiency myelopathy: Perspectives on the clinical biochemistry of vitamin B12.

Beginning with a case report of nitrous oxide (N2O)-induced B12 deficiency myelopathy, this article reviews the clinical biochemistry of vitamin B12, and examines the pathogenetic mechanisms by which B12 deficiency leads to neurologic damage, and how this damage is potentiated by N2O exposure. The article systematically examines the available experimental data relating to the two main coenzyme mechanisms that are usually suggested in clinical articles, particularly the deficient methylation hypothesis. The article demonstrates that neither of these mechanisms is fully consistent with the available data. The article then presents a novel mechanism based on new data from the neuroimmunology basic science literature which suggests that the pathogenesis of B12 deficiency myelopathy may not be related to its role as a coenzyme, but rather to newly discovered functions of B12 in regulating cytokines and growth factors.

Mecobalamin.

BACKGROUND: Mecobalamin, one of the coenzyme forms of vitamin B(12), acts as an important cofactor in the activities of B(12)-dependent methyltransferases. Since the discovery of mecobalamin, it has been applied mainly in the treatment of hyperhomocysteinaemia and peripheral neuropathy. However, there is still lack of a systemic review on the clinical administration of mecobalamin and its potential mechanism. OBJECTIVE: To review the mechanism, clinical efficacy and safety of mecobalamin in the treatment of hyperhomocysteinaemia and peripheral neuropathy. METHODS: First, the potential mechanism, pharmacokinetics and metabolism of mecobalamin were clarified. In addition, the clinical administration including efficacy, safety and tolerability of mecobalamin as monotherapy or combined therapy in the treatment of hyperhomocysteinaemia and peripheral neuropathy were also detailed. RESULTS/CONCLUSIONS: Although both monotherapy and combined therapy can lower plasma/serum homocysteine levels and improve the neuropathic symptoms, combined therapy with other B vitamins seems to be more effective. However, more precise, double-blind and randomised control studies are necessary to confirm the efficacy of mecobalamin on hyperhomocysteinaemia, peripheral neuropathy interaction, and cardiovascular, neurological and osteoporotic mortality or morbidity.

The nitric oxide scavenger cobinamide profoundly improves survival in a Drosophila melanogaster model of bacterial sepsis.

Septic shock has an extremely high mortality rate, with approximately 200,000 people dying from sepsis annually in the U.S. The high mortality results in part from severe hypotension secondary to high serum NO concentrations. Reducing NO levels should be beneficial in sepsis, but NOS inhibitors have had a checkered history in animal models, and one such agent increased mortality in a clinical trial. An alternative approach to reduce NO levels in sepsis is to use an NO scavenger, which should leave sufficient free NO for normal physiological functions. Using a well-established model of bacterial sepsis in Drosophila melanogaster, we found that cobinamide, a B(12) analog and an effective NO scavenger in vitro, dramatically improved fly survival. Cobinamide augmented the effect of an antibiotic and was beneficial even in immune-deficient flies. Cobinamide's mechanism of action appeared to be from reducing NO levels and improving cardiac function.

Radical enzymes in anaerobes.

This review describes enzymes that contain radicals and/or catalyze reactions with radical intermediates. Because radicals irreversibly react with dioxygen, most of these enzymes occur in anaerobic bacteria and archaea. Exceptions are the families of coenzyme B(12)- and S-adenosylmethionine (SAM)-dependent radical enzymes, of which some members also occur in aerobes. Especially oxygen-sensitive radical enzymes are the glycyl radical enzymes and 2-hydroxyacyl-CoA dehydratases. The latter are activated by an ATP-dependent one-electron transfer and act via a ketyl radical anion mechanism. Related enzymes are the ATP-dependent benzoyl-CoA reductase and the ATP-independent 4-hydroxybenzoyl-CoA reductase. Ketyl radical anions may also be generated by one-electron oxidation as shown by the flavin-adenine-dinucleotide (FAD)- and [4Fe-4S]-containing 4-hydroxybutyryl-CoA dehydratase. Finally, two radical enzymes are discussed, pyruvate:ferredoxin oxidoreductase and methane-forming methyl-CoM reductase, which catalyze their main reaction in two-electron steps, but subsequent electron transfers proceed via radicals.

Anaerobic chlorophyll isocyclic ring formation in Rhodobacter capsulatus requires a cobalamin cofactor.

The isocyclic ring of bacteriochlorophyll (BChl) is formed by the conversion of Mg-protoporphyrin monomethyl ester (MPE) to protochlorophyllide (PChlide). Similarities revealed by blast searches with the putative anaerobic MPE-cyclase BchE suggested to us that this protein also uses a cobalamin cofactor. We found that vitamin B(12) (B(12))-requiring mutants of the bluE and bluB genes of Rhodobacter capsulatus, grown without B(12), accumulated Mg-porphyrins. Laser desorption/ionization time-of-flight (LDI-TOF) MS and NMR spectroscopy identified them as MPE and its 3-vinyl-8-ethyl (mvMPE) derivative. An in vivo assay was devised for the cyclase converting MPE to PChlide. Cyclase activity in the B(12)-dependent mutants required B(12) but not protein synthesis. The following reaction mechanism is proposed for this MPE-cyclase reaction. Adenosylcobalamin forms the adenosyl radical, which leads to withdrawal of a hydrogen atom and formation of the benzylic-type 13(1)-radical of MPE. Withdrawal of an electron gives the 13(1)-cation of MPE. Hydroxyl ion attack on the cation gives 13(1)-hydroxy-MPE. Withdrawal of three hydrogen atoms leads successively to 13(1)-keto-MPE, its 13(2)-radical, and cyclization to PChlide.

The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli.

The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria.

The synthesis of a pyridyl analog of adenosylcobalamin and its coenzymic function in the diol dehydratase reaction.

A novel analog of adenosylcobalamin in which 5,6-dimethylbenzimidazole and D-ribose moieties of the nucleotide loop are replaced by pyridine and the trimethylene group, respectively, was synthesized and examined for coenzymic function. The coordination of pyridine to the cobalt atom in this analog was stronger than that of 5,6-dimethylbenzimidazole in the corresponding homolog. The adenosyl form of pyridyl analog served as partially active coenzyme for diol dehydratase. The kcat/Km values calculated from the initial velocity indicate that this analog is a better coenzyme than the 5,6-dimethylbenzimidazolyl or imidazolyl counterpart. However, the reaction with the pyridyl analog as coenzyme was accompanied with a concomitant inactivation during catalysis, with a kcat/Kinact value 50-100 times lower than that for adenosylcobalamin or the 5,6-dimethylbenzimidazolyl analog. Therefore, it can be concluded that the 5,6-dimethylbenzimidazole moiety of adenosylcobalamin is important for continuous progress of a catalytic cycle by protecting the reactive intermediates from side reactions.

Essential role of adenosylcobalamin in leucine synthesis from beta-leucine in the domestic chicken.

This study was designed to investigate a postulated relationship between vitamin B-12 and leucine metabolism in mature domestic chickens. Plasma amino acid analysis revealed the presence of beta-leucine at a concentration of 60 to 80 mumol/l. After 425 d on a vitamin B-12-deficient diet, plasma beta-leucine was 133% higher (P less than 0.06) and plasma leucine and methionine lower (P less than 0.03) than values in plasma from hens fed a diet adequate in vitamin B-12. Branched-chain-amino-acid aminotransferase (EC 2.6.1.42) (BCAT) activity was not enhanced by vitamin B-12 deprivation (P greater than 0.05). In contrast to leucine, beta-leucine was not utilized as substrate by BCAT for the formation of alpha-ketoisocaproate. Kidney extracts possessed leucine 2,3-aminomutase (EC 5.4.3.7) (LAM) activity, as evidenced by enhanced conversion of beta-leucine to alpha-leucine in the presence of adenosylcobalamin. LAM activity could not be demonstrated in liver or muscle extract, while leucine formation by pancreas extract was negligible. These data represent the first evidence of the presence of the amino acid beta-leucine in chicken plasma. In addition, the data support vitamin B-12-dependent leucine synthesis from beta-leucine in the chicken and highlight the kidney's role in leucine synthesis.

Altered cobalamin distribution in rat hepatomas and in the livers of rats treated with diethylnitrosamine.

The distribution of cobalamin cofactors was investigated in the livers and tumors of rats bearing transplanted Morris 7777 or 7800 hepatomas, in the livers of rats treated with the hepatocarcinogen diethylnitrosamine, and in normal rats. There was a significant increase in the proportion of methylcobalamin both in livers and tumors from rat bearing the hepatomas 7777 and 7800 compared to the proportion of methylcobalamin in the livers of normal rats. The total cobalamin content of the hepatomas was significantly lower than that of host or control livers. Similarly, the total cobalamin content of the livers from the tumor-bearing rats was less than that in control animals. The administration to rats of an acute dose of diethylnitrosamine led to an 84% increase in the hepatic concentration of methylcobalamin. Chronic administration of diethylnitrosamine slightly increased the hepatic methylcobalamin concentration, but this was not statistically significant. Liver weight was reduced, and the hepatic content of total cobalamin fell to 55% of that in control animals.