Isolation of two novel corrinoid proteins from acetate-grown Methanosarcina barkeri.
Two corrinoid proteins with molecular sizes of 480 and 29 kDa are stably methylated by [2-14C]acetate-derived intermediates in cell extracts of aceticlastic Methanosarcina barkeri when methylreductase is inhibited by the addition of bromoethanesulfonic acid. Both 14CH3-proteins have been isolated to near homogeneity and found to be abundant soluble proteins. The larger protein possesses two subunits, of 41.4 and 30.4 kDa, in an equimolar ratio, suggesting an alpha 6 beta 6 conformation with six bound methylated corrinoids per 480-kDa molecule. The 29-kDa protein is a monomer in solution and possesses only one methylated corrinoid. All methyl groups on both proteins are photolabile, but the methylated corrinoid bound to the 29-kDa protein undergoes photolysis at a higher rate than that bound to the 480-kDa protein. The two proteins possess discrete N termini and do not appear to be forms of the same protein in equilibrium. Neither protein has an Fe4S4 cluster, and both have UV-visible spectra most similar to that of a base-on methylated corrinoid. A previously identified methylated protein, designated the unknown A 14CH3-protein, copurifies with the 480-kDa protein and has the same subunit composition. The methyl groups of both isolated 14CH3-proteins are converted to methane in cell extracts. The methylated proteins that accumulate in extracts in the presence of bromoethanesulfonic acid are demethylated by the addition of coenzyme M. Both isolated proteins are abundant novel corrinoid proteins that can methylate and be methylated by intermediates of the methanogenic pathway.
Kremer
J D
JD
Department of Microbiology, Ohio State University, Columbus 43210.
Cao
X
X
Krzycki
J
J
D000085:Acetates; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D045728:Corrinoids; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000032:analysis; D017021:Methanosarcina barkeri / Q000737:chemistry* / Q000378:metabolism; D008745:Methylation; D010782:Photolysis; D013053:Spectrophotometry; D014805:Vitamin B 12 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000085:Acetates; D001426:Bacterial Proteins; D045728:Corrinoids; D007501:Iron; D014805:Vitamin B 12