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Abstract

[Fe]-hydrogenase (Hmd) catalyzes the reduction of methenyl-H4MPT+ to methylene-H4MPT using H2 as electron donor in the hydrogenotrophic methanogenic pathway. The production of Hmd was upregulated when the cell was grown under Ni-limiting environment. Hmd is composed of homodimer; the active sites are located at the cleft formed by the N-terminal domain and central domain. The N-terminal domain binds an iron-guanylylpyridinol (FeGP) cofactor, which is prosthetic group of this enzyme. The FeGP cofactor is composed of a low spin FeII ligated with two CO, an acyl-C and pyridinol-N; in addition, Cys-S and a solvent are bound to the iron site in the enzyme. The pyridinol ring is substituted with GMP moiety and two methyl groups. Genome analysis indicated that there are seven conserved genes which is named hcg gene cluster containing hcgAG and hmd genes. Therefore, it was predicted that the hcg cluster is responsible for biosynthesis of the FeGP cofactor. From the hcg genes sequences, we could not deduce the function of the proteins. However, using the “structure to function” strategy and biochemical assays, we could identify the function of some Hcg proteins. In this thesis, I describe the function of HcgC based on crystal structure and biochemical analyses. The isotope-labeling experiment indicated that the C3 methyl group comes from methionine, probably via S-adenosylmethionine (SAM). Structure comparisons of HcgC with other proteins suggested similarity of HcgC to SAM-dependent methyltransferases. Co-crystallization of HcgC and SAM revealed that SAM binds to the active site of HcgC. Docking simulation with a possible methyl-acceptor pyridinol suggested that the binding site of the pyridinol. The predicted substrate pyridinol was chemically synthesized and the enzyme activity was determined. The structure of the HcgC-reaction product was determined by NMR, which confirmed that HcgC transfer the methyl group from SAM to C3 of pyridinol. In order to analyze the catalytic mechanism of HcgC, co-crystallizaiton of HcgC, pyridinol, SAM or SAH was performed. The substrate binding site structure showed that seven water molecules connected pyridinol to protein. The only interaction of pyridinol with amino acid side chain was Thr179-OH. The C3 of pyridinol was close to the sulfur of SAH. In the crystal structure, there was no amino acid, which functions as general base of the typical methyl-transfer reaction. We proposed that the water molecules stabilize the deprotonated form of pyridinol by resonance effect, which increases the nucleophilicity of C3. Mutation analysis supported the essential contribution of the water molecules.