Microbial protein structure

Methane is an end product of the anaerobic degradation of organic materials. This combustible gas is useful as a fuel but also it is a potential greenhouse gas (1). Huge amounts of methane are produced by methanogenic archaea on Earth. Increasing anthropogenic methane production could become a global issue. We are interested in the enzymes involved in methanogenesis from H₂ and CO₂ (Figure 1). In the hydrogenotrophic methanogenic pathway, many unique enzymes are involved (2). These methanogenic enzymes contain novel cofactors and use unique coenzymes as the substrates. My group is mainly working on three major projects on the methanogenic enzymes:

- Structure and function of the enzymes involved in hydrogenotrophic methanogenesis.

- Catalytic mechanism of [Fe]-hydrogenase.

- Biosynthesis of the [Fe]-hydrogenase cofactor.

Structure and function of the enzymes involved in methanogenesis

In this project, my group intensively collaborates with Ulrich Ermler (X-ray crystallography) from the Max Planck Institute of Biophysics in Frankfurt. We have elucidated the structure of formyl-methanofuran dehydrogenase (Fwd) (3), formyl-methanofuran:tetrahydromethanopterin (H₄MPT) formyltransferase (4-6), methenyl-H₄MPT⁺-cyclohydrolase (Mch) (7, 8), F₄₂₀-dependent methylene-H₄MPT dehydrogenase (Mtd) (9, 10), H₂-forming methylene-H₄MPT dehydrogenase ([Fe]-hydrogenase or Hmd) (11-14), methylene-H₄MPT reductase (Mer) (15, 16), the MtrA subunit of membrane-spanning methyl-H₄MPT:coenzyme M methyltransferase complex (MtrA–H) (17), methyl-coenzyme M reductase (Mcr) (18-22),  Heterodisulfide-reductase/[NiFe]-hydrogenase complex (Hdr/Mvh) (23), F₄₂₀-reducing [NiFe]-hydrogenase (Frh) (24-26), F₄₂₀H₂ oxidase (FprA) (27, 28), and F₄₂₀-dependent alcohol dehydrogenase (29) from methanogenic archaea. In addition, we solved the structures of acetoacetyl-CoA thiolase/HMG-CoA synthase complex (30) and HMG-CoA reductase (31) from a methanogenic archaeon, F₄₂₀:NADP oxidoreductase from a sulfate-reducing archaeon (32), NADP dependent methylene-H₄MPT dehydrogenase (33) and formyltransferase-hydrolase complex (34) from a methylotrophic bacterium, and methyl-coenzyme M reductase from a methylotrophic archaeon (35, 36). As examples, the structure and function of Fwd and Mvh-Hdr are described below.

Formyl-methanofuran dehydrogenase (Fwd). Hydrogenotrophic methanogens start the metabolism by an ATP-independent CO₂ fixation process. This reaction is catalyzed by formyl-methanofuran dehydrogenase (Fwd), which fixes CO₂ on the methanofuran coenzyme to form formyl-methanofuran (Figure 1, Reaction 1). We solved the crystal structure of the Fwd complex (3). Fwd is organized in a tetramer of heterohexamer, Fwd(ABCDFG)₄,which contains 46 [4Fe-4S] clusters (Figure 2A). Most biological CO₂ fixation metabolisms start with trapping CO₂ as a carboxy group, which is followed by a reduction. In contrast, Fwd uses a different strategy, where CO₂ is firstly reduced to formate, which is then fixed on methanofuran to generate formyl-methanofuran (Figure 2B). The subunit FwdB, which is homologous to formate dehydrogenase, contains a tungstopterin active site and a channel for CO₂ entry. Electrons are transferred through the [4Fe-4S] clusters to the tungstopterin for the CO₂ reduction to formate. The formate formed will be sent to the next active site through a 43-Å-long tunnel. FwdA is a homolog of amidohydrolase and catalyzes the condensation of formate to methanofuran. One of the unexpected features of the 800-kDa complex is the electronic wire composed of 46 [4Fe-4S]-clusters (Figure 2A). The arrangement of [4Fe-4S] clusters functions as an electron relay, and may synchronize the four tungstopterin active sites over 206 Å.

Heterodisulfide-reductase/[NiFe]-hydrogenase complex (Hdr/Mvh). The final step of the methanogenic pathway is catalyzed by Mcr (Figure 1, Reaction 8), which generates two products: methane and the heterodisulfide (CoM-S-S-CoB). The reduced coenzyme M and B are regenerated from CoM-S-S-CoB by the reaction catalyzed by heterodisulfide-reductase/[NiFe]-hydrogenase complex (HdrABC-MvhAGD) (Figure 1, Reaction 9). HdrA is the catalytic center of a new mode of energy-coupling mechanism “flavin-based electron bifurcation”. In this process, 4 electrons are separated into 2 electrons with lower energy and 2 electrons with higher energy for reduction of heterodisulfide and ferredoxin, respectively. The reduced ferredoxin gives high-energy electrons to CO₂, which is catalyzed by formyl-methanofuran dehydrogenases (Fwd) described above. To explore the catalytic mechanism of the HdrABC-MvhAGD complex, we solved the crystal structure of the HdrABC-MvhAGD complex from a methanogen Methanothermococcus thermolithotrophicus (23). The crystal structure allowed us to draw the plausible electron transfer pathways from the hydrogenase to the heterodisulfide reductase modules in this enzyme complex (Figure 3). Location of flavin and the conserved amino-acid residues at the flavin binding-site in HdrA supported the proposed function of flavin in the electron-bifurcation mechanism. HdrA homologs are found in various enzyme complexes in numerous microorganisms. The HdrA structure of the HdrABC-MvhAGD complex serves as the prototype of this protein family. In addition, in the HdrB subunit, we found a new type of iron-sulfur cluster, noncubane [4Fe-4S] cluster, which is bound to the amino-acid residues at the CCG motifs. The CCG motif is found in 2000 other proteins but the structure and function was unknown. Cryo-intermediate-trapping experiments indicated that the noncubane iron-sulfur clusters performed the reduction of the CoM-S-S-CoB.

        Recently, we solved the structure of a 3-Mda complex of formate dehydrogenase (Fdh), Hdr, and formylmethanofuran dehydrogenase (Fmd) in a hexamer of the Fdh-Hdr-Fmd dimer from Methanospirillum hungatei (63). This megacomplex enzyme catalyzes the reduction of CO₂ to formyl-MFR with low potential electrons provided by the flavin-based electron bifurcation mechanism in Hdr using electrons from formate and the reduced form of F₄₂₀. The low-potential electron-output domain of Hdr and the CO₂-reducing domain in Fmd are linked by electron chains of the FmdF polyferredoxin.

The catalytic mechanism of [Fe]-hydrogenase

Molecular hydrogen and proton are used as electron donor or acceptor, respectively, in the metabolism of many anaerobic microorganisms. Hydrogenases catalyze the reversible reaction (37). The most prominent hydrogenases are [NiFe]-hydrogenase and [FeFe]-hydrogenase. [Fe]-hydrogenase (Hmd) is not related to either [NiFe]-hydrogenase or [FeFe]-hydrogenase (38). This third type of hydrogenases contains a mononuclear iron cofactor, iron-guanylylpyridinol (FeGP) cofactor cartooned in Figure 4 and 5. This enzyme catalyzes the reversible dehydrogenation of methylene-H₄MPT to methenyl- H₄MPT⁺. We solved the atomic-resolution (1.06 Å) structure of [Fe]-hydrogenase bound with substrate, methenyl-H₄MPT⁺, in which the activated enzyme is ready for H₂-binding and -activation at the mono-Fe site (11). Compared to the substrate-free [Fe]-hydrogenase, the closed active [Fe]-hydrogenase displays a different coordination structure of the Fe center. In the activated enzyme, the active-site cleft closed and the water ligand of the Fe site is dissociated from the Fe center, which results in the penta-coordinated Fe complex (Figure 4). The removal of the water ligand makes the open site accessible for the H₂ binding. The structural change was supported by Mössbauer and infrared spectroscopic analyses. The deprotonated 2-OH group of the FegP cofactor acts as catalytic base, where H₂-binding to Fe, H₂-cleavage and finally hydride transfer proceeds smoothly at the active site. In the crystal structure, the substrate is highly distorted. Based on structural analyses and kinetic studies (13, 39-44), we illustrate a catalytic cycle of the [Fe]-hydrogenase (2, 11).

We constructed active [Fe]-hydrogenase holoenzymes with a mimetic iron-pyridinol model compound and the apoenzyme in collaboration with the group of Xile Hu (Ecole polytechnique fédérale de Lausanne, EPFL) (45). The mimetic cofactor lacks the bulky GMP nucleotide and two methyl substituents compared to the native cofactor. Despite the modifications, the turnover frequency of the reconstituted enzyme was 1–2 s⁻¹, which is much higher than that of synthetic hydrogenation catalysts. This is the first construction of an active metalloenzyme using a truncated metal cofactor. In addition, we showed that constructing the enzyme using a mimetic cofactor containing a 2-hydroxy pyridine group restores activity, while an analogous experiment with a 2-methoxy-pyridine complex leads to an inactive enzyme. These findings support a mechanism in which the 2-hydroxy group is deprotonated before serving as an internal base for heterolytic H₂ cleavage. Our results open a door in the design of new mimetic active-site cofactors for hydrogenation and the production of H₂, an important future energy currency. We have recently constructed a [Mn]-hydrogenase using a Mn model complex (46).

Biosynthesis of the [Fe]-hydrogenase cofactor

Because of the unique structural and functional features of the FeGP cofactor (11, 14, 47-53), its biosynthesis is of great interest in chemistry and biology. Based on retro-synthetic analysis and stable-isotope-labeling data, we proposed a pathway for the FeGP cofactor biosynthesis involving various reactions for pyridinol formation, pyridinol methylation, and iron center formation (54, 55). In many methanogenic archaea, the hmd genes ([Fe]-hydrogenase structural genes) are clustered with hmd-co-occurring genes (hcgA–G), which suggests that the hcgA–G genes are involved in biosynthesis of the FeGP cofactor (37, 55). To analyze the function of the hcg genes, we employed a “structure–to-function” strategy.

HcgB. We first elucidated the function of the HcgB protein using this approach (56). Our comparative genomic studies revealed a structural relationship between HcgB and nucleotide triphosphatases, which was not ascertainable at the sequence level. Superimposed structures of HcgB and nucleotide triphosphatase indicated that the nucleotide triphosphate-binding cleft of nucleoside triphosphatase is similar to that of HcgB. However, a crucial aspartate, the base catalyst for hydrolysis of nucleoside triphosphates, is exchanged in HcgB with a glycine. Therefore, we predicted that HcgB has the ability to bind and activate nucleoside triphosphate, but lacks nucleoside triphosphatase activity. These findings sparked the idea that HcgB might catalyze a guanylyltransferase reaction to conjugate the pyridinol ring to GMP. To test this hypothesis, we used GTP and pyridinol model substrates in the presence of HcgB from Methanocaldococcus jannaschii in biosynthetic model reactions to form the corresponding guanylyl-pyridinol products. Using NMR, we identified 3,6-dimethyl-2,4-dihydroxypyridine-GMP as the model reaction product. In the cocrystal structure of HcgB from M. jannaschii guanylylpyridinol (GP) precursor was located inside the predicted substrate-binding cleft. Kinetic studies indicated that HcgB catalyzes formation of GP from the pyridinol precursors and GTP (55, 56).

HcgC. The pyridinol ring of the FeGP cofactor is substituted with two methyl groups, an acylmethyl group, and GMP. Crystal structure of HcgC from Methanocaldococcus jannaschii was solved and we found that HcgC was structurally similar to S-adenosylmethionine (SAM)-dependent methyltransferases, which suggested the potential function as a methyltransferase (57). Previous isotope-labeling experiment indicated that the 3-methyl group of the pyridinol ring was obtained from methionine most probably via SAM. The predicted precursor (methyl acceptor) of the HcgC reaction was chemically synthesized in collaboration with Xile Hu (EPFL). The methylation reaction catalyzed by HcgC was confirmed by experiments using the synthesized compound (57). Furthermore, we clarified the unique catalytic mechanism, which use water molecules to activate the pyridinol substrate (58).

HcgD. This enzyme is a homolog of the ubiquitous Nif3-like protein family proteins, which contain dinuclear metal center. HcgD contains a dinuclear iron center. One of the irons is removed by chelating reagents. This finding suggests that HcgD might have an iron-trafficking function in biosynthesis of the FeGP cofactor (59).

HcgE. The primary structure of HcgE resembles that of E1-like ubiquitin-activating enzymes (E1 enzymes). E1 enzymes catalyze adenylylation of the C-terminal end of ubiquitin(-like) proteins using ATP as adenylyl group donor. The structure of the co-crystal of HcgE with ATP and GP revealed that GP binds to the active site pocket of HcgE, where the carboxy group points towards the  a-phosphate of ATP (60). This finding suggested that HcgE catalyzes adenylylation of the 6-carboxymethyl group of GP. The HcgE-catalyzed reaction was kinetically confirmed.

HcgF. This enzyme is not similar in primary structure to any proteins of known function. Co-crystal structures indicated that the HcgF homodimer has two GP-binding sites. One of two GP molecules is partially bound to HcgF by covalent bonding of the carboxyl group of GP to Cys9-thiolate (60). Such thioester bonding of the C-terminal carboxyl group and a cysteine-thiolate is observed in the ubiquitin-activation process. Our hypothesis based on chemical precedents is that the thioester reacts with an iron species to form the acyl and thiolate ligands to iron of the cofactor.

Based on the results, a biosynthetic sequence of the FeGP cofactor is proposed. A pyridinol precursor is synthesized from β-alanine (or aspartate) and 2,3-dihydroxy-4-oxo-pentanoate like compound, which were predicted by retrosynthesis. The 3 position of pyridinol is methylated and the 4 position is conjugated with GMP to form GP. The 6-carboxy group of GP pyridinol ring is activated by adenylylation reaction with ATP and activated via formation of a thioester bond in HcgF, which suggests Fe-acyl formation via a nucleophilic substitution reaction to allow the release of Cys9 of HcgF as thiolate.

HcgA. The function of HcgA was studied using an in vitro biosynthesis assay with cell extract from M. maripaludis mutants containing a reaction cooktail. The first known pyridinol precursor of FeGP cofactor is biosynthesized by the reaction catalyzed by HcgA (64, 65). HcgA is a radical SAM enzyme and forms the pyridinol precursor from an unknown precursor in the cell extract of M. maripaludis.

HcgG. The in vitro biosynthetic assays indicated that HcgG catalyzes the reactions involved in completing the iron complex structure of FeGP cofactor; biosynthesis of CO and acyl ligands and insertion of Fe (64, 65). The radical SAM reaction is involved in the HcgG catalyzed reaction.

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