title: Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina.
abstract: Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Deep
authors.initials: D
authors.lastname: Chatterjee
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, the German Cancer Consortium (DKTK), Heidelberg D-69210, Germany, and the German Cancer Research Center (DKFZ), Heidelberg D-69210, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Denis
authors.initials: D
authors.lastname: Kudlinzki
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Linhard
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, the German Cancer Consortium (DKTK), Heidelberg D-69210, Germany, and the German Cancer Research Center (DKFZ), Heidelberg D-69210, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Krishna
authors.initials: K
authors.lastname: Saxena
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, the German Cancer Consortium (DKTK), Heidelberg D-69210, Germany, and the German Cancer Research Center (DKFZ), Heidelberg D-69210, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schieborr
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, the German Cancer Consortium (DKTK), Heidelberg D-69210, Germany, and the German Cancer Research Center (DKFZ), Heidelberg D-69210, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Santosh L
authors.initials: SL
authors.lastname: Gande
authors.affiliation: the Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Jan Philip
authors.initials: JP
authors.lastname: Wurm
authors.affiliation: the Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Wöhnert
authors.affiliation: Institute of Biochemistry, Biocenter, and.
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Rogov
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: the Institute for Molecular Biosciences.
authors.forename: Heinz D
authors.initials: HD
authors.lastname: Osiewacz
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany, sridhar@nmr.uni-frankfurt.de.
authors.forename: Sridhar
authors.initials: S
authors.lastname: Sreeramulu
authors.affiliation: From the Institute for Organic Chemistry and Chemical Biology, the German Cancer Consortium (DKTK), Heidelberg D-69210, Germany, and the German Cancer Research Center (DKFZ), Heidelberg D-69210, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe University, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany, schwalbe@nmr.uni-frankfurt.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Schwalbe
mesh_terms: D001703:Biophysics; D018360:Crystallography, X-Ray; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D048468:Podospora / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012436:S-Adenosylmethionine / Q000378:metabolism*
chemical_list: D005419:Flavonoids; D005656:Fungal Proteins; C040015:myricetin; D012436:S-Adenosylmethionine; D008780:Methyltransferases
keywords: PaMTH1; Podospora anserina; aging; cancer; methyltransferase; myricetin; nuclear magnetic resonance (NMR); reactive oxygen species (ROS); x-ray crystallography