Reconstitution of Monomethylamine:Coenzyme M methyl transfer with a corrinoid protein and two methyltransferases purified from Methanosarcina barkeri.
Methanogenesis from methylamines requires the intermediate methylation of 2-mercaptoethanesulfonate (CoM). In vitro reconstitution of CoM methylation with monomethylamine was achieved with three purified proteins: a monomethylamine corrinoid protein (MMCP), the "A" isozyme of methylcobamide:CoM methyltransferase (MT2-A), and a newly isolated protein termed monomethylamine methyltransferase (MMAMT).MMAMT is a 170-kDa protein with 52-kDa subunits. The MMAMT polypeptide was rate-limiting for methyl transfer until at a 2-fold molar excess over MMCP. MMAMT is a monomethylamine:MMCP methyltransferase, since methylation of MMCP required MMAMT but not MT2-A. MMCP and MMAMT formed a complex detectable by size exclusion high pressure liquid chromatography. Methyl group transfer from methyl-MMCP to CoM was mediated by MT2-A, since methyl iodide:CoM methyl transfer by MMCP and MT2-A did not require MMAMT. MT2-M, an isozyme of MT2-A, was inactive in MMCP-dependent methyl transfer. Immunodepletion of MMCP from the extract inhibited CoM methylation with monomethylamine but not dimethylamine. Purified MMCP reconstituted activity in immunodepleted extracts. These results show that MMCP is the major corrinoid protein for methanogenesis from monomethylamine detectable in extracts and that it interacts with two methyltransferases. MMAMT functions as a MMA:MMCP methyltransferase, while MT2-A functions as a methyl-MMCP:CoM methyltransferase.
Burke
S A
SA
Department of Microbiology, Ohio State University, Columbus, Ohio 43210, USA.
Krzycki
J A
JA
D001426:Bacterial Proteins / Q000378:metabolism*; D015080:Mesna / Q000378:metabolism*; D017020:Methanosarcina / Q000201:enzymology*; D008744:Methylamines / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*
D001426:Bacterial Proteins; D008744:Methylamines; C027451:methylamine; D008780:Methyltransferases; D015080:Mesna