Fujishiro, TakashiBai, LipingXu, TaoXie, XiulanSchick, MichaelKahnt, JoergRother, MichaelHu, XileErmler, UlrichShima, Seigo2016-10-182016-10-182016-10-18201610.1002/anie.201604352https://infoscience.epfl.ch/handle/20.500.14299/130359WOS:000383372700033Previous 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.cofactorsenzymeshydrogenasesmethyltransferasesprotein structurestext::journal::journal article::research article