title: Identification of HcgC as a SAM-Dependent Pyridinol Methyltransferase in [Fe]-Hydrogenase Cofactor Biosynthesis.
abstract: Previous retrosynthetic and isotope-labeling studies have indicated that biosynthesis of the iron guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase requires a methyltransferase. This hypothetical enzyme covalently attaches the methyl group at the 3-position of the pyridinol ring. We describe the identification of HcgC, a gene product of the hcgA-G cluster responsible for FeGP cofactor biosynthesis. It acts as an S-adenosylmethionine (SAM)-dependent methyltransferase, based on the crystal structures of HcgC and the HcgC/SAM and HcgC/S-adenosylhomocysteine (SAH) complexes. The pyridinol substrate, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol, was predicted based on properties of the conserved binding pocket and substrate docking simulations. For verification, the assumed substrate was synthesized and used in a kinetic assay. Mass spectrometry and NMR analysis revealed 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol as the reaction product, which confirmed the function of HcgC.
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße 10, 35043, Marburg, Germany.|Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, Shimo-ohkubo 255, Sakura-ku, Saitama, 338-8570, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Fujishiro
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße 10, 35043, Marburg, Germany.
authors.forename: Liping
authors.initials: L
authors.lastname: Bai
authors.affiliation: Institute of Chemical Science and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), ISIC-LSCI, BCH 3305, 1015, Lausanne, Switzerland.
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
authors.affiliation: Department of Chemistry, Philipps Universität Marburg, Hans-Meerwein-Straße, 35032, Marburg, Germany.
authors.forename: Xiulan
authors.initials: X
authors.lastname: Xie
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße 10, 35043, Marburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schick
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße 10, 35043, Marburg, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Kahnt
authors.affiliation: Institut für Mikrobiologie, Technische Universität Dresden, 01062, Dresden, Germany.
authors.forename: Michael
authors.identifier: 0000-0002-2306-8783
authors.initials: M
authors.lastname: Rother
authors.affiliation: Institute of Chemical Science and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), ISIC-LSCI, BCH 3305, 1015, Lausanne, Switzerland.
authors.forename: Xile
authors.initials: X
authors.lastname: Hu
authors.affiliation: Max-Planck-Institut für Biophysik, Max-von-Laue-Straße 3, 60438, Frankfurt/Main, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Ermler
authors.affiliation: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße 10, 35043, Marburg, Germany. shima@mpi-marburg.mpg.de.|PRESTO, Japan, Science and Technology Agency, JST, Saitama, 332-0012, Japan. shima@mpi-marburg.mpg.de.
authors.forename: Seigo
authors.identifier: 0000-0003-1872-8705
authors.initials: S
authors.lastname: Shima
keywords: cofactors; enzymes; hydrogenases; methyltransferases; protein structures