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J. Biol. Chem., Vol. 275, Issue 37, 29053-29060, September 15, 2000
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From the 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.
Reconstitution of Dimethylamine:Coenzyme M Methyl Transfer with a
Discrete Corrinoid Protein and Two Methyltransferases Purified from
Methanosarcina barkeri*
§,
Department of Microbiology, The
Ohio State University, Columbus, Ohio 43210 and the ¶ Department
of Biochemistry and Molecular Biology, Uniformed Services University of
the Health Sciences, Bethesda, Maryland 20814
*
This work was supported by United States Department of
Energy Grant DE-FG-02-91ER20042 (to J. A. K.) and by National Science Foundation Grant MCB-9905068 (to D. A. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
614-292-1578; Fax: 614-292-8120; E-mail: Krzycki.1@osu.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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