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 ...

Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5-deoxyadenosylcobalamin by MMACHC are 11.7 ؎ 0.2 and 0.174 ؎ 0.006 h ؊1 at 20°C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B 12 -dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.

The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B 12 ). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5′-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using

Microtubule affinity regulating kinase 4 (MARK4) is a Ser/Thr kinase belonging to AMPK-like family, has recently become an important drug target against cancer and neurodegenerative disorders. In this study, we have evaluated different natural dietary polyphenolics including rutin, quercetin, ferulic acid, hesperidin, gallic acid and vanillin as MARK4 inhibitors. All compounds are primarily binds to the active site cavity of MARK4. In silico observations were further complemented by the fluorescence-binding studies and isothermal titration calorimetry (ITC) measurements. We found that rutin and vanillin bind to MARK4 with a reasonably high affinity. ATPase and tau-phosphorylation assay further suggesting that rutin and vanillin inhibit the enzyme activity of MARK4 to a great extent. Cell proliferation, ROS quantification and Annexin-V staining studies are clearly providing sufficient evidences for the apoptotic potential of rutin and vanillin. In conclusion, rutin and vanillin may be considered as potential inhibitors for MARK4 and further exploited to design novel therapeutic molecules against MARK4 associated diseases.

The hadBC and hadI genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2-hydroxyisocaproyl-CoA dehydratase HadBC and its activator HadI. The activated enzyme catalyses the dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA in the pathway of leucine fermentation. The extremely oxygen-sensitive homodimeric activator as well as the heterodimeric dehydratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehydratase. The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP, a process similar to that observed with the unrelated nitrogenase. The thus activated dehydratase was separated from the activator and ATP; it catalyzed about 10(4) dehydration turnovers until the enzyme became inactive. Adding activator, ATP, MgCl(2), dithionite and dithioerythritol reactivated the enzyme. This is the first demonstration with a 2-hydroxyacyl-CoA dehydratase that the catalytic electron is recycled after each turnover. In agreement with this observation, only substoichiometric amounts of activator (dehydratase/activator = 10 mol/mol) were required to generate full activity.