Subtitle: [Fe]-hydrogenase: Role of iron-sulfur bonding in holoenzyme assembly and in FeGP-cofactor biosynthesis
[Fe]-hydrogenase catalyzes the reversible transfer of a hydride from H2to methenyl-tetrahydromethanopterin which is an intermediary step in methanogenesis from H2 and CO2. The enzyme contains one iron per active site. The iron is associated with a unique iron-guanylylpyridinol (FeGP) cofactor. In many methanogens, the [Fe]-hydrogenase structural gene (hmd) is clustered with hmd-co-occurring genes (hcgA-G), which have been shown to be involved in FeGP cofactor biosynthesis. In previous studies, we have already identified the function of five of the hcg gene-products (HcgB, HcgC, HcgD, HcgE and HcgF) using structure to function strategies and biochemical assays. HcgB catalyzed guanylyl-transfer from GTP to the 4-hydroxypyridinol. HcgC is a SAM-dependent methyltransferase; HcgD is a putative iron chaperone. HcgE catalyzes the adenylylation of the carboxy group of a 6-carboxymethyl-guanylylpyridinol precursor. Subsequently the product of HcgE reacts with Cys9 of HcgF yielding a thioester and AMP. Based on chemical precedents, we propose that the thioester reacts with an iron species forming the acyl and thiolate ligands. In the next three years, we would like to identify the function of the two remaining Hcg proteins (HcgA and HcgG), both of which are predicted to be iron-sulfur proteins. For this purpose, we will - as before - employ structure to function strategies, in vitro biosynthesis and metabolite analysis of hcg knock-out mutants.
Period: 01.09.2016 - 31.08.2019