In Vitro Biosynthesis of the FeGP Cofactor of the [Fe]-Hydrogenase
Life requires many challenging chemical reactions, which are enabled by metallocofactors of enzymes. One such reaction is splitting of molecular hydrogen (H2) by hydrogenases, which are classified based on their cofactors as [NiFe]-, [FeFe]- or [Fe]-hydrogenases. These enzymes are of special interes...
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|Summary:||Life requires many challenging chemical reactions, which are enabled by metallocofactors of enzymes. One such reaction is splitting of molecular hydrogen (H2) by hydrogenases, which are classified based on their cofactors as [NiFe]-, [FeFe]- or [Fe]-hydrogenases. These enzymes are of special interest due to their potential application in solutions for future energy storage. The [Fe]-hydrogenase reversibly catalyzes the heterolytic cleavage of H2 into a proton and a hydride. The latter is subsequent transferred to methenyl-tetrahydromethanopterin, which is involved in the hydrogenotrophic methanogenesis. The [Fe]-hydrogenase contains the iron-guanylylpyridinol (FeGP) cofactor as prosthetic group of this enzyme. This cofactor consists of a low-spin Fe(II), which is coordinated by two CO, one cysteine sulfur and one bidentate acyl-methylene-pyridinol ligands. In this work, the biosynthesis of the FeGP cofactor was investigated. The cofactor is presumably synthesized by the reactions of the HcgA G proteins. In previous studies, the functions of HcgB–F have already been partially analyzed. To investigate the missing function of HcgA and HcgG, as well as to confirm the nature of the precursors of the FeGP cofactor, we developed a method of synthesizing the FeGP cofactor in vitro using a mixture of defined and undefined compounds in combination with proteins. The in vitro biosynthesis solution contains the [Fe] hydrogenase apoenzyme, a possible precursor, e.g. 6 carboxyl-methylene-3,5-dimethyl-4-guanylyl-2-pyridinol (compound 3), ATP/Mg2+, S-adenosyl methionine, dithiothreitol and sodium dithionite, Hcg proteins, and cell extract from a Methanococcus maripaludis Δhcg mutant that lacks endogenous [Fe] hydrogenase activity. Based on the results of the in vitro biosynthesis, we confirmed the structure of the pyridinol precursors, which were predicted based on investigations of the Hcg proteins. Importantly, we confirmed that a carboxyl group of these pyridinol precursors is converted into the acyl ligand of the FeGP cofactor. In addition, we found that the in vitro biosynthesis requires reducing equivalents, which can be generated from H2 or formate. We then observed requirements of some small cellular components such as a possible CO precursor and an electron carrier for the biosynthesis reactions. We demonstrated that CO gas can also be incorporated into the CO ligands. Furthermore we confirmed that the reaction of HcgE generates a proposed adenylylated compound 3 in the presence of ATP, which was also necessary for the in vitro biosynthesis, and that the compound 3 is bound to HcgF. Moreover, we showed that HcgA catalyzes the biosynthesis of the initial pyridinol precursor, 6-carboxyl-methylene-4-hydroxyl-5-methyl-2-pyridinol. Further we demonstrated that HcgG catalyzes the biosynthesis of the FeGP cofactor from the guanylylpyridinol precursor 3 and the components from the cell extract of methanogens (see PhD thesis of F. Arriaza). In addition, in this thesis, I discuss the production of the FeGP cofactor from compound 3 in a fully defined protein mixture containing HcgE, HcgG and small components from the cell extract of M. maripaludis based on some preliminary results.|
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