Seigo Shima

Hokkaido University, Sapporo, Hokkaidō, Japan

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Publications (87)574.41 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: HcgD, a homolog of the ubiquitous Nif3-like protein family, is found in a gene cluster involved in the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase. The presented crystal structure and biochemical analyses indicated that HcgD has a dinuclear iron-center, which provides a pronounced binding site for anionic ligands. HcgD contains a stronger and a weaker bound iron; the latter being removable by chelating reagents preferentially in the oxidized state. Therefore, we propose HcgD as an iron chaperon in FeGP cofactor biosynthesis, which might also stimulate investigations on the functionally unknown but physiologically important eukaryotic Nif3-like protein family members.
    FEBS letters. 06/2014;
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    ABSTRACT: The reversible redox reaction between coenzyme F420 and H2 to F420H2 is catalyzed by a F420-reducing [NiFe]-hydrogenase (FrhABG) which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high resolution cryo electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but different because of the truncation of ca. 160 residues which results in similar but modified H2- and proton- transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of a N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F420-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°' ≈ -400mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.
    Journal of molecular biology. 05/2014;
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    ABSTRACT: Consortia of anaerobic methanotrophic (ANME) archaea and delta-proteobacteria anaerobically oxidize methane coupled to sulfate reduction to sulfide. The metagenome of ANME-1 archaea contains genes homologous to genes otherwise only found in methanogenic archaea, and transcription of some of these genes in ANME-1 cells has been shown. We now have heterologously expressed three of these genes in Escherichia coli, namely those homologous to genes for formylmethanofuran:tetrahydromethanopterin formyltransferase (Ftr), methenyltetrahydromethanopterin cyclohydrolase (Mch), and coenzyme F420-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd), and have characterized the overproduced enzymes with respect to their coenzyme specificity and other catalytic properties. The three enzymes from ANME-1 were found to catalyze the same reactions and with similar specific activities using identical coenzymes as the respective enzymes in methanogenic archaea, the apparent Km for their substrates being in the same concentration range. The results support the proposal that anaerobic oxidation of methane to CO2 in ANME involves the same enzymes and coenzymes as CO2 reduction to methane in methanogenic archaea. Interestingly, the activity of Mch and the stability of Mtd from ANME-1were found to be dependent on the presence of 0.5-1.0 M potassium phosphate, which suggested that ANME-1 archaea contain high concentrations of lyotropic salts, presumably as compatible solutes.
    Environmental Microbiology 04/2014; · 6.24 Impact Factor
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    ABSTRACT: Coenzyme F430 is a nickel hydrocorphinoid, and is the prosthetic group of methyl-coenzyme M reductase that catalyzes the last step of the methanogenic reaction sequence and its reversed reaction for anaerobic methane oxidation by ANME. As such, function-specific compound analysis has the potential to reveal the microbial distribution and activity associated with methane production and consumption in natural environments and, in particular, in deep subsurface sediments where microbiological and geochemical techniques are restricted. Herein, we report the development of a technique for high-sensitivity analysis of F430 in environmental samples, including paddy soils, marine sediments, microbial mats, and an anaerobic groundwater. The lower detection limit of F430 analysis by liquid chromatography / mass spectrometry is 0.1 fmol, which corresponds to 6 × 102 to 1 × 104 cells of methanogens. F430 concentrations in these natural environmental samples range from 6 to 44 nmol g-1 and are consistent with the methanogenic archaeal biomass estimated by microbiological analyses.
    Analytical Chemistry 03/2014; · 5.70 Impact Factor
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    ABSTRACT: HcgD and HcgD bind by X-ray crystallography (View interaction)
    01/2014;
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    ABSTRACT: [Fe]-Hydrogenase requires the iron guanylylpyridinol (FeGP) cofactor for activity. The function of HcgB, an enzyme in the biosynthesis of the FeGP cofactor, was predicted by structural genomics and confirmed by model reactions and various analytical methods: HcgB catalyzes the terminal guanylyltransferase reaction for the formation of guanylylpyridinol. GMP=guanosine monophosphate.
    Angewandte Chemie International Edition 11/2013; 52(48):12555-8. · 11.34 Impact Factor
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    ABSTRACT: Inhibition mechanism: Isocyanides strongly inhibit [Fe]-hydrogenase. X-ray crystallography and X-ray absorption spectroscopy revealed that the isocyanide binds to the trans position, versus the acyl carbon of the Fe center, and is covalently bound to the pyridinol hydroxy oxygen. These results also indicated that the hydroxy group is essential for H2 activation.
    Angewandte Chemie International Edition 07/2013; · 11.34 Impact Factor
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    ABSTRACT: Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI:http://dx.doi.org/10.7554/eLife.00218.001.
    eLife Sciences 01/2013; 2:e00218.
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    ABSTRACT: One reaction step of the biosynthesis of [Fe]‐hydrogenase‐cofactor is elucidated by S. Shima et al. in their Communication on page 12555 ff. A structural genomics approach, in combination with model reactions and thorough product analysis by X‐ray crystallography of the protein–product complexes, revealed that HcgB is the enzyme that catalyzes guanylylpyridinol formation from a 2,4‐dihydroxypyridine derivative and guanosine triphosphate.
    Angewandte Chemie International Edition 01/2013; 52(48). · 11.34 Impact Factor
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    ABSTRACT: Ein Reaktionsschritt der Biosynthese des [Fe]‐Hydrogenase‐Cofaktors wurde von S. Shima et al. in ihrer Zuschrift auf S. 12787 ff. aufgeklärt. Mithilfe struktureller Genomik in Kombination mit Modellreaktionen und gründlicher Produktanalyse durch Röntgenkristallographie von Protein‐Produkt‐Komplexen wurde gefunden, dass HcgB das Enzym ist, das die Guanylylpyridinolbildung aus einem 2,4‐Dihydroxypyridinderivat und Guanosintriphosphat katalysiert.
    Angewandte Chemie 01/2013; 125(48).
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    ABSTRACT: Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) at marine gas seeps is performed by archaeal-bacterial consortia that have so far not been cultivated in axenic binary or pure cultures. Knowledge about possible biochemical reactions in AOM consortia is based on metagenomic retrieval of genes related to those in archaeal methanogenesis and bacterial sulfate reduction, and identification of a few catabolic enzymes in protein extracts. Whereas the possible enzyme for methane activation (a variant of methyl-coenzyme M reductase, Mcr) was shown to be harboured by the archaea, enzymes for sulfate activation and reduction have not been localized so far. We adopted a novel approach of fluorescent immunolabelling on semi-thin (0.3-0.5 μm) cryosections to localize two enzymes of the SR pathway, adenylyl : sulfate transferase (Sat; ATP sulfurylase) and dissimilatory sulfite reductase (Dsr) in microbial consortia from Black Sea methane seeps. Both Sat and Dsr were exclusively found in an abundant microbial morphotype (c. 50% of all cells), which was tentatively identified as Desulfosarcina/Desulfococcus-related bacteria. These results show that ANME-2 archaea in the Black Sea AOM consortia did not express bacterial enzymes of the canonical sulfate reduction pathway and thus, in contrast to previous suggestions, most likely cannot perform canonical sulfate reduction. Moreover, our results show that fluorescent immunolabelling on semi-thin cryosections which to our knowledge has been so far only applied on cell tissues, is a powerful tool for intracellular protein detection in natural microbial associations.
    Environmental Microbiology 09/2012; · 6.24 Impact Factor
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    ABSTRACT: Methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) cyclohydrolase (Mch) catalyzes the interconversion of methenyl-H(4)MPT(+) and formyl-H(4)MPT in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria. To understand the catalytic mechanism of this reaction, we kinetically characterized site-specific variants of Mch from Archaeoglobus fulgidus (aMch) and determined the X-ray structures of the substrate-free aMch(E186Q), the aMch:H(4)MPT complex, and the aMch(E186Q):formyl-H(4)MPT complex. (Formyl-)H(4)MPT is embedded inside a largely preformed, interdomain pocket of the homotrimeric enzyme with the pterin and benzyl rings being oriented nearly perpendicular to each other. The active site is primarily built up by the segment 93:95, Arg183 and Glu186 that either interact with the catalytic water attacking methenyl-H(4)MPT(+) or with the formyl oxygen of formyl-H(4)MPT. The catalytic function of the strictly conserved Arg183 and Glu186 was substantiated by the low enzymatic activities of the E186A, E186D, E186N, E186Q, R183A, R183Q, R183E, R183K, and R183E-E186Q variants. Glu186 most likely acts as a general base. Arg183 decisively influences the pK(a) value of Glu186 and the proposed catalytic water mainly by its positive charge. In addition, Glu186 appears to be also responsible for product specificity by donating a proton to the directly neighbored N(10) tertiary amine of H(4)MPT. Thus, N(10) becomes a better leaving group than N(5) which implies the generation of N(5)-formyl-H(4)MPT. For comparison, methenyltetrahydrofolate (H(4)F) cyclohydrolase produces N(10)-formyl-H(4)F in an analogous reaction. An enzymatic mechanism of Mch is postulated and compared with that of other cyclohydrolases.
    Biochemistry 09/2012; 51(42):8435-43. · 3.38 Impact Factor
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    ABSTRACT: [Fe]-hydrogenase catalyzes the reversible hydride transfer from H(2) to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H(2) and CO(2) in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe(II) is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme's cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of (13)C and (2)H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO(2). The metabolic origin of the two CO-ligands was CO(2) rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO(2) via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of (13)CO, the two CO-ligands and the acyl group became (13)C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized.
    Journal of the American Chemical Society 02/2012; 134(6):3271-80. · 10.68 Impact Factor
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    ABSTRACT: The anaerobic oxidation of methane (AOM) with sulphate, an area currently generating great interest in microbiology, is accomplished by consortia of methanotrophic archaea (ANME) and sulphate-reducing bacteria. The enzyme activating methane in methanotrophic archaea has tentatively been identified as a homologue of methyl-coenzyme M reductase (MCR) that catalyses the methane-forming step in methanogenic archaea. Here we report an X-ray structure of the 280 kDa heterohexameric ANME-1 MCR complex. It was crystallized uniquely from a protein ensemble purified from consortia of microorganisms collected with a submersible from a Black Sea mat catalysing AOM with sulphate. Crystals grown from the heterogeneous sample diffract to 2.1 Å resolution and consist of a single ANME-1 MCR population, demonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in complex with coenzyme M and coenzyme B, indicating the same substrates for MCR from methanotrophic and methanogenic archaea. Differences between the highly similar structures of ANME-1 MCR and methanogenic MCR include a F(430) modification, a cysteine-rich patch and an altered post-translational amino acid modification pattern, which may tune the enzymes for their functions in different biological contexts.
    Nature 11/2011; 481(7379):98-101. · 38.60 Impact Factor
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    ABSTRACT: [Fe]-hydrogenase catalyzes the reversible heterolytic cleavage of H(2) and stereo-specific hydride transfer to the substrate methenyltetrahydromethanopterin in methanogenic archaea. This enzyme contains a unique iron guanylylpyridinol (FeGP) cofactor as a prosthetic group. It has recently been proposed-on the basis of crystal structural analyses of the [Fe]-hydrogenase holoenzyme-that the FeGP cofactor contains an acyl-iron ligation, the first one reported in a biological system. We report here that the cofactor can be reversibly extracted with acids; its exact mass has been determined by electrospray ionization Fourier transform ion cyclotron resonance mass-spectrometry. The measured mass of the intact cofactor and its gas-phase fragments are consistent with the proposed structure. The mass of the light decomposition products of the cofactor support the presence of acyl-iron ligation. Attenuated total reflection infrared spectroscopy of the FeGP cofactor revealed a band near wave number 1700 cm(-1), which was assigned to the C=O (double bond) stretching mode of the acyl-iron ligand.
    Dalton Transactions 11/2011; 41(3):767-71. · 3.81 Impact Factor
  • Seigo Shima, Ulrich Ermler
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    ABSTRACT: [Fe]-hydrogenase functions in the methanogenic pathway of hydrogenotrophic methanogenic archaea. It catalyzes thereversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT and a proton by transferring a hydride ion to the proR position of the C14a carbon atom of methylene-H4MPT. This third type of hydrogenase contains a unique iron–guanylylpyridinol (FeGP) cofactor, in which the iron atom is ligated by one cysteine sulfur atom, two CO groups, one solvent molecule, and an sp2-hybridized nitrogen atom and an acyl carbon atom from the pyridinol ring. Three globular folding units of this protein form two clefts that serve as substrate-binding and active sites and that can be open or closed. Structural data are presented for the apoenzyme in a closed form, the holoenzyme (enzyme with the FeGP cofactor), the C176A holoenzyme, and the binary C176A holoenzyme-methylene-H4MPT complex in an open form. A closed and potentially active binary complex has been reliably modeled on the basis of the open binary complex and the closed apoenzyme. In this model, the iron center sits near the Re face of the imidazolidine ring of the substrate. The iron ligation site trans to the acyl carbon atom is next to the C14a carbon atom and is therefore considered to be the H2 binding site.
    ChemInform 06/2011; 42(25).
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    ABSTRACT: Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.
    Environmental Microbiology 03/2011; 13(5):1370-9. · 6.24 Impact Factor
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    ABSTRACT: [Fe]-hydrogenase is one of the three types of hydrogenases. This enzyme is found in many hydrogenotrophic methanogenic archaea and catalyzes the reversible hydride transfer from H(2) to methenyl-H(4)MPT(+) in methanogenesis from H(2) and CO(2). The enzyme harbors a unique iron-guanylyl pyridinol (FeGP) cofactor as a prosthetic group. Here, we describe the purification of [Fe]-hydrogenase from Methanothermobacter marburgensis, the isolation of the FeGP cofactor from the native holoenzyme, and the reconstitution of [Fe]-hydrogenase from the isolated FeGP cofactor and the heterologously produced apoenzyme.
    Methods in enzymology 01/2011; 494:119-37. · 1.90 Impact Factor
  • Seigo Shima, Kenichi Ataka
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    ABSTRACT: [Fe]-Hydrogenase catalyzes the reversible activation of H(2). CO and CN(-) inhibit this enzyme with low affinity (K(i)≅0.1 mM) by binding to the iron site of the bound iron-guanyrylpyridinol cofactor. We report here that isocyanides, which are formally isoelectronic with CO and CN(-), strongly inhibit [Fe]-hydrogenase (K(i) as low as 1 nM). The [NiFe]- and [FeFe]-hydrogenases tested were not inhibited by isocyanides. UV-Vis and infrared spectra revealed that the isocyanides bind to the iron center of [Fe]-hydrogenase. The inhibition kinetics are in agreement with the proposed catalytic mechanism, including the open/closed conformational change of the enzyme.
    FEBS letters 01/2011; 585(2):353-6. · 3.54 Impact Factor
  • Angewandte Chemie International Edition 12/2010; 49(51):9917-21. · 11.34 Impact Factor

Publication Stats

2k Citations
574.41 Total Impact Points

Institutions

  • 2014
    • Hokkaido University
      • Institute of Low Temperature Science
      Sapporo, Hokkaidō, Japan
  • 2005–2014
    • Max Planck Institute for Terrestrial Microbiology
      Marburg, Hesse, Germany
  • 2004–2012
    • Max Planck Institute for Marine Microbiology
      • Department of Biogeochemistry
      Bremen, Bremen, Germany
  • 1997–2012
    • Max Planck Institute of Biophysics
      • Department of Molecular Membrane Biology
      Frankfurt, Hesse, Germany
  • 2005–2011
    • Max Planck Institute for Empirical Aesthetics
      Frankfurt, Hesse, Germany
  • 1995–2008
    • Philipps-Universität Marburg
      • Fachbereich Biologie
      Marburg an der Lahn, Hesse, Germany
  • 2002
    • Goethe-Universität Frankfurt am Main
      • Institut für Biophysik
      Frankfurt am Main, Hesse, Germany
  • 1998
    • University of Cologne
      • Institute of Organic Chemistry
      Köln, North Rhine-Westphalia, Germany