title: Molecular characterization of methanogenic N(5)-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase.
abstract: Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
authors.affiliation: Max-Planck-Institut für Biophysik, Max-von-Laue-Straße 3, D-60438 Frankfurt am Main, Germany.
authors.forename: Vikrant
authors.initials: V
authors.lastname: Upadhyay
authors.affiliation: Max-Planck-Institut für Biophysik, Max-von-Laue-Straße 3, D-60438 Frankfurt am Main, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Ceh
authors.affiliation: Goethe-Universität Frankfurt am Main, Institut für Biochemie, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Tumulka
authors.affiliation: Goethe-Universität Frankfurt am Main, Institut für Biochemie, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
authors.affiliation: Institut für Physikalische und Theoretische Chemie, Goethe-Universität Frankfurt am Main, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Hoffmann
authors.affiliation: Max-Planck-Institut für Biophysik, Max-von-Laue-Straße 3, D-60438 Frankfurt am Main, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Langer
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, Germany; PRESTO, Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: shima@mpi-marburg.mpg.de.
authors.forename: Seigo
authors.initials: S
authors.lastname: Shima
authors.affiliation: Max-Planck-Institut für Biophysik, Max-von-Laue-Straße 3, D-60438 Frankfurt am Main, Germany. Electronic address: ulrich.ermler@biophys.mpg.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Ermler
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D017013:Methanobacteriaceae / Q000737:chemistry* / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D011622:Pterins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D003067:Coenzymes; D008565:Membrane Proteins; D011622:Pterins; C042416:5,6,7,8-tetrahydromethanopterin; D008780:Methyltransferases
keywords: Dimethyl maleic anhydride; LILBID-MS; Membrane protein complex; Methanogenesis; N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase; SEC-MALS