mesna and cobamamide

Formaldehyde conversion into methyl-coenzyme M involves (a) reaction of the substrate with 5,6,7,8-tetrahydromethanopterin (H4MPT) giving 5,10-methylene-H4MPT, followed by its reduction to 5-methyl-H4MPT and (b) transfer of the methyl group from the latter compound to coenzyme M. The reactions were studied in a resolved system from Methanobacterium thermoautotrophicum strain delta H. The first part (a) of the reactions was catalyzed by the 55% ammonium sulfate supernatant of cell-free extracts. The methyltransferase step (b) was dependent on an oxygen-sensitive enzyme, called methyltransferase a (MTa). Isolation of MTa was achieved by gel filtration on Sephacryl S-400. MTa was a high-molecular-weight complex of at least 2000 kDa and between 900 to 1500 kDa when purified in the absence and presence of the detergent CHAPS, respectively. The enzyme consisted of 100 kDa units composed of three subunits in an alpha beta gamma configuration with apparent molecular masses of 35, 33 and 31 kDa, respectively. The corrinoid, 5-hydroxybenzymidazolyl cobamide (B12HBI, Factor III) copurified with MTa and the latter contained 2 nmol B12HBI per mg protein. B12HBI present in MTa could be methylated under the appropriate conditions by 5-methyl-H4MPT. These findings suggest that the corrinoid is a prosthetic group of MTa. MTa may be homologous to the corrinoid membrane protein purified before from M. thermoautotrophicum strain Marburg (Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Eur. J. Biochem. 171, 589-597).

Topics: Cobamides; Mesna; Methanobacterium; Methyltransferases; Pterins

Corrins activated the methylreductase system from Methanobacterium bryantii three- to fivefold in extracts resolved from low-molecular-weight factors. Corrins did not substitute for ATP and component B, which were also required for maximal activity. The concentration of diaquacobinamides required for one-half maximal activity was 1 microM. The concentrations of cyanocobalamin, methylcobalamin, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, and 5'-deoxyadenosylcobinamide required for one-half maximal activity were between 4 and 7 microM. Deoxyadenosylcobalamin was nearly inactive. Activation was independent of thiols, coenzyme M, and ATP. Activation was also observed after partial purification of the methylreductase system by agarose column chromatography. Corrins were required in catalytic concentrations, methylcobalamin was not required, and methanogenesis was enzymatic. Corrin activation of the methylreductase is a novel effect on methanogenesis. However, the physiological significance of the corrin activation is uncertain.

Topics: Cobamides; Corrinoids; Enzyme Activation; Euryarchaeota; Kinetics; Mesna; NADP; Oxidoreductases; Sulfhydryl Compounds; Vitamin B 12