Arriaza-Gallardo, Francisco J.Schaupp, SebastianZheng, Yu-CongAbdul-Halim, Mohd FaridPan, Hui-JieKahnt, JoergAngelidou, GeorgiaPaczia, NicoleHu, XileCosta, KyleShima, Seigo2022-12-192022-12-192022-12-192022-11-1710.1002/anie.202213239https://infoscience.epfl.ch/handle/20.500.14299/193355WOS:000888920100001In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis Delta hcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the Delta hcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.Chemistry, MultidisciplinaryChemistryacyl ligandsbiosynthesisfegp cofactorradical s-adenosyl methionine enzymes[fe]-hydrogenasefree hydrogenase hmdacyl-iron ligationcrystal-structureactive-sitemethanogenic archaeaidentificationtext::journal::journal article::research article