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Zusammenfassung:
Most methanogenic archaea reduce CO2 to methane using molecular hydrogen (H2) as the electron donor. Specific metalloenzymes called hydrogenases catalyze oxidation of H2 or hydride transfer from H2 to provide electrons to the metabolism. Under nickel-limiting conditions, production of [Fe]-hydrogenase (Hmd) is up-regulated, and it functions as one of the major hydrogenases in hydrogenotrophic methanogens. Hmd catalyzes the reversible reduction of methenyl-tetrahydromethanopterin (methenyl-H4MPT+) to methylene-H4MPT by transferring a hydride from H2. This enzyme possesses a unique metallocofactor, the iron-guanylylpyridinol (FeGP) cofactor, in its active site. The FeGP cofactor contains an Fe-center, a pyridinol ring, and a GMP moiety. The iron center is coordinated with two CO ligands, one cysteine-sulfur, the nitrogen of the pyridinol ring, and an acyl-carbon of one of the substituents of the pyridinol ring. Biosynthesis of this cofactor requires the product of at least seven genes, which are named hmd co-occurring genes (hcgA-G). HcgC is a SAM-dependent methyltransferase that methylates the 3-position of 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (precursor 1), to make precursor 2. HcgB is a GTP-dependent enzyme that guanylylates precursor 2 to produce a guanylylpyridinol (GP or precursor 3). HcgE is an ATP-dependent enzyme that adenylylates 3 and activates the carboxyl group. The adenylylated product is predicted to bind to HcgF forming a thioester bond with a cysteine residue. HcgD is a possible iron-trafficking protein. In this work, I elucidated the function of the remaining Hcg proteins (HcgA and HcgG). I expressed the hcgA gene in Escherichia coli and purified an active form of HcgA. By using a newly developed in vitro biosynthesis assay (Schaupp and Arriaza et al. 2022. Angew. Chem. Int. Ed. 61, e202200994), I determined HcgA as a radical SAM enzyme that produces precursor 1 from an unknown compound. Since HcgG tends to form inclusion bodies in the Escherichia coli cells, I expressed the hcgG gene homologously in Methanococcus maripaludis, and purified an active form of HcgG. In vitro biosynthesis experiments suggested that the HcgG is a radical SAM enzyme that catalyzes multiple catalytic reactions: (1) production of CO from an unknown cellular component, (2) biosynthesis of the CO ligands, (3) formation of the acyl-ligand, and (4) assembly of the FeGP cofactor including incorporation of Fe2+. In addition, I investigated the reconstitution of the holoenzyme in vitro from the apoenzyme and the isolated FeGP cofactor. Based on the kinetic and structural analyses, I proposed a mechanism of the FeGP cofactor binding to the protein. Furthermore, I studied the function of the Hmd paralog (HmdII) by mutation analyses. These experiments suggested that HmdII is overproduced in the M. maripaludis strain (∆frh) lacking F420-reducing [NiFe]-hydrogenase activity and that HmdII negatively regulates the production of the FeGP cofactor.
