N5-methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein.
An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N5-methyltetrahydromethanopterin (CH3-H4MPT) to coenzyme M (H-S-CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H2/CO(2)-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH3-S-CoM formation from CH3-H4MPT (apparent Km = 50 microM) and H-S-CoM (apparent Km = 250 microM) of approximately 0.6 mumol.min-1.mg protein-1. For activity the presence of Ti(III) citrate (apparent Km = 15 microM) and of ATP (apparent Km = 30 microM) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH3-H4MPT in the absence of H-S-CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH3-H4MPT to H-S-CoM in the absence of ATP and Ti(III). Demethylation in the presence of H-S-CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.
Fischer
R
R
Laboratorium für Mikrobiologie des Fachbereichs Biologie, Philipps-Universität Marburg, Federal Republic of Germany.
Gärtner
P
P
Yeliseev
A
A
Thauer
R K
RK
D019605:Euryarchaeota / Q000201:enzymology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D015080:Mesna / Q000378:metabolism*; D008780:Methyltransferases / Q000032:analysis* / Q000378:metabolism; D011622:Pterins / Q000378:metabolism*
D008565:Membrane Proteins; D011622:Pterins; D008780:Methyltransferases; C048755:tetrahydromethanopterin S-methyltransferase; D015080:Mesna