title: Reconstitution of dimethylamine:coenzyme M methyl transfer with a discrete corrinoid protein and two methyltransferases purified from Methanosarcina barkeri.
abstract: Methyl transfer from dimethylamine to coenzyme M was reconstituted in vitro for the first time using only highly purified proteins. These proteins isolated from Methanosarcina barkeri included the previously unidentified corrinoid protein MtbC, which copurified with MtbA, the methylcorrinoid:Coenzyme M methyltransferase specific for methanogenesis from methylamines. MtbC binds 1.0 mol of corrinoid cofactor/mol of 24-kDa polypeptide and stimulated dimethylamine:coenzyme M methyl transfer 3.4-fold in a cell extract. Purified MtbC and MtbA were used to assay and purify a dimethylamine:corrinoid methyltransferase, MtbB1. MtbB1 is a 230-kDa protein composed of 51-kDa subunits that do not possess a corrinoid prosthetic group. Purified MtbB1, MtbC, and MtbA were the sole protein requirements for in vitro dimethylamine:coenzyme M methyl transfer. An MtbB1:MtbC ratio of 1 was optimal for coenzyme M methylation with dimethylamine. MtbB1 methylated either corrinoid bound to MtbC or free cob(I)alamin with dimethylamine, indicating MtbB1 carries an active site for dimethylamine demethylation and corrinoid methylation. Experiments in which different proteins of the resolved monomethylamine:coenzyme M methyl transfer reaction replaced proteins involved in dimethylamine:coenzyme M methyl transfer indicated high specificity of MtbB1 and MtbC in dimethylamine:coenzyme M methyl transfer activity. These results indicate MtbB1 demethylates dimethylamine and specifically methylates the corrinoid prosthetic group of MtbC, which is subsequently demethylated by MtbA to methylate coenzyme M during methanogenesis from dimethylamine.
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Ferguson
authors.forename: N
authors.initials: N
authors.lastname: Gorlatova
authors.forename: D A
authors.initials: DA
authors.lastname: Grahame
authors.forename: J A
authors.initials: JA
authors.lastname: Krzycki
mesh_terms: D004123:Dimethylamines / Q000378:metabolism*; D015080:Mesna / Q000378:metabolism*; D017021:Methanosarcina barkeri / Q000201:enzymology*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*
chemical_list: D004123:Dimethylamines; C034516:dimethylamine; D008780:Methyltransferases; D015080:Mesna