Isolation of a 5-hydroxybenzimidazolyl cobamide-containing enzyme involved in the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in Methanobacterium thermoautotrophicum.
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).
Kengen
S W
SW
Department of Microbiology, Faculty of Science, University of Nijmegen, The Netherlands.
Daas
P J
PJ
Duits
E F
EF
Keltjens
J T
JT
van der Drift
C
C
Vogels
G D
GD
D003038:Cobamides / Q000737:chemistry*; D015080:Mesna / Q000378:metabolism*; D017014:Methanobacterium / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D011622:Pterins / Q000378:metabolism*
D003038:Cobamides; D011622:Pterins; C042416:5,6,7,8-tetrahydromethanopterin; D008780:Methyltransferases; C048755:tetrahydromethanopterin S-methyltransferase; C000913:cobamamide; D015080:Mesna