mesna and 5-6-7-8-tetrahydromethanopterin

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

A new enzyme, tetrahydromethanopterin methyltransferase, which catalyzes the transfer of methyl groups from methyl-tetrahydromethanopterin to 2-mercaptoethane-sulfonate, has been found in the methane synthesizing complex of Methanobacterium thermoautotrophicum. The enzyme is oxygen sensitive and has a well defined pH optimum at pH 6.7. There was no methyl group transfer when the enzyme was heated to 100 degrees for 5 min. The product of the forward reaction, methyl-CoM, was positively identified by TLC and high voltage paper electrophoresis. The demethylation of methyl-CoM, in the absence of methane synthesis, was dependent on the addition of H4MPT which suggests that the enzyme reaction is reversible.

Topics: Euryarchaeota; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Mesna; Methane; Methyltransferases; Pterins

A fraction previously isolated from acid-treated supernatant fraction of Methanobacterium thermoautotrophicum by DEAE-Sephadex chromatography [Sauer, Mahadevan & Erfle (1984) Biochem. J. 221, 61-97] which was absolutely required for methane synthesis, has been separated into two compounds, tetrahydromethanopterin (H4MPT) and an as-yet-unidentified cofactor we call 'cytoplasmic cofactor'. H4MPT was identified by its u.v. spectrum and by 13C- and 1H-n.m.r. spectroscopy. The reduction of 2-(methylthio)ethanesulphonic acid (CH3-S-CoM) to methane by the membrane fraction from M. thermoautotrophicum was completely dependent on the addition of cytoplasmic cofactor. Methane synthesis from CO2, however, was only partially dependent on cofactor addition, and 57% of the original activity was retained in its absence. The kinetics of 14C labelling were consistent with the scheme methyl-H4MPT----CH3-S-CoM----methane, as has been proposed. This is the first time that direct experimental evidence has been presented to show that the proposed methyl transfer from H4MPT to coenzyme M (HS-CoM) actually occurs.

Topics: Cell Membrane; Chromatography, Ion Exchange; Euryarchaeota; Kinetics; Magnetic Resonance Spectroscopy; Mercaptoethanol; Mesna; Methane; Pterins; Spectrophotometry, Ultraviolet; Surface-Active Agents