Yoshitsugu Shiro

Hokkaido University, Sapporo, Hokkaidō, Japan

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Publications (222)910.85 Total impact

  • Akihiro Doi · Hiro Nakamura · Yoshitsugu Shiro · Hiroshi Sugimoto ·
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    ABSTRACT: ChrA is a response regulator (RR) in the two-component system involved in regulating the degradation and transport of haem (Fe–porphyrin) in the pathogen Corynebacterium diphtheriae . Here, the crystal structure of full-length ChrA is described at a resolution of 1.8 Å. ChrA consists of an N-terminal regulatory domain, a long linker region and a C-terminal DNA-binding domain. A structural comparison of ChrA with other RRs revealed substantial differences in the relative orientation of the two domains and the conformation of the linker region. The structural flexibility of the linker could be an important feature in rearrangement of the domain orientation to create a dimerization interface to bind DNA during haem-sensing signal transduction.
    Acta Crystallographica Section F: Structural Biology Communications 08/2015; 71(Pt 8):966-71. DOI:10.1107/S2053230X15009838
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    ABSTRACT: L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme catalyzing the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe2+-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain still elusive. In the present study, the O2 inserting reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using heme-modification approach, which enabled to draw quantitative correlation between the inductive electronic effects of the heme-substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in the preparation of reconstituted PaTDO with synthetic hemes, which were different in the inductive electron-withdrawing nature of the heme-substituents at the 2- and 4-positions. An increase in the electron-withdrawing power of the heme-substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme-substituents, the lesser the electron density on the heme-iron. The decrease in the electron density of the heme-iron resulted in the higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the O2 dissociation from the ferrous-oxy intermediate. This result was interpreted due to a lesser  back-donation from the heme-iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme-substituents, implying that the higher electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.
    Biochemistry 05/2015; 54(23). DOI:10.1021/acs.biochem.5b00048 · 3.02 Impact Factor
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    ABSTRACT: The direct hydroxylation of alkanes under mild conditions is a key issue in catalytic chemistry that addresses an increasing number of industrial and economic requirements. Cytochrome P450s are monooxygenases that are capable of oxidizing less reactive C–H bonds; however, wild-type P450s are unavailable for many important nonnative substrates such as gaseous alkanes. Here, we report the enhanced hydroxylation activities and crystallographic evidence for the role of decoy molecules in wild-type P450BM3-catalyzed hydroxylation of gaseous ethane and propane by using the next generation of decoy molecule. A cocrystal structure of P450BM3 and a decoy molecule reveals that an N-perfluoroacyl amino acid (decoy molecule) partially occupies the substrate-binding site of P450BM3. This binding of the decoy re-forms the active site pocket to allow the accommodation of small substrates and simultaneously influences the formation of compound I species by expelling water molecules from the active site.
    ACS Catalysis 01/2015; 5(1):150-159. DOI:10.1021/cs501592f · 9.31 Impact Factor
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    ABSTRACT: Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4-Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. Arg190Ala and Arg81Ala mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg190 and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM-derivatives with anti-osteoclast activity.
    Journal of Biological Chemistry 09/2014; 289(47). DOI:10.1074/jbc.M114.598391 · 4.57 Impact Factor
  • Yoshitsugu Shiro · Takehiko Tosha · Erina Terasaka · Hiroshi Sugimoto ·

    Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e5. DOI:10.1016/j.bbabio.2014.05.125 · 5.35 Impact Factor
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    ABSTRACT: Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2 O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.7 Å, to elucidate structure-function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO-bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH3 -CH=N-OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ~0.5 Å increase in the heme/non-heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton-pumping activity in cNOR, because redox-coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non-heme Fe distance even in the bulky ligand-bound form of cNOR (~4.5 Å) than the heme/Cu distance in CCO (~5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N-N coupling to produce a hyponitrite intermediate for the generation of N2 O. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 07/2014; 82(7). DOI:10.1002/prot.24492 · 2.63 Impact Factor
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    ABSTRACT: Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
    Biochimica et Biophysica Acta 07/2014; 1837(7). DOI:10.1016/j.bbabio.2014.02.017 · 4.66 Impact Factor
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    ABSTRACT: A sulfur-modified gold-supported palladium material (SAPd) has been developed bearing palladium nanoparticles on its surface. Herein, we report the for the first time the use of SAPd to affect a Pd-nanoparticle-catalyzed 1,7-Pd migration reaction for the synthesis of benzotriazoles via C-H bond activation. The resulting benzotriazoles were evaluated in terms of their inhibitory activity towards indoleamine 2,3-dioxygenase.
    ChemInform 06/2014; 79(13). DOI:10.1021/jo5009838
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    ABSTRACT: The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasAp ) can capture several synthetic metal complexes other than heme. The crystal structures of HasAp harboring synthetic metal complexes revealed only small perturbation of the overall HasAp structure. An inhibitory effect upon heme acquisition by HasAp bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasAp bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasAp as an iron source.
    Angewandte Chemie International Edition 03/2014; 53(11):2862-6. DOI:10.1002/anie.201307889 · 11.26 Impact Factor

  • Angewandte Chemie 03/2014; 126(11). DOI:10.1002/ange.201401090
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    ABSTRACT: Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands.
    Journal of inorganic biochemistry 03/2014; 135C:20-27. DOI:10.1016/j.jinorgbio.2014.02.011 · 3.44 Impact Factor
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    ABSTRACT: The O2-binding carboxylate-bridged diiron site in DcrH-Hr was engineered in an effort to perform the H2O2-dependent oxidation of external substrates. A His residue was introduced near the diiron site in place of a conserved residue, Ile119. The I119H variant promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H2O2.
    Chemical Communications 01/2014; 50(26). DOI:10.1039/c3cc48108e · 6.83 Impact Factor
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    ABSTRACT: A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.
    Inorganic Chemistry 11/2013; 52(22). DOI:10.1021/ic401632x · 4.76 Impact Factor
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    ABSTRACT: FixL is a heme-based oxygen-sensing histidine kinase that induces expression of nitrogen fixation genes under hypoxic conditions. Oxygen binding to heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, inactivating autophosphorylation activity. Although FixL also can bind other diatomic ligands such as CO, the CO-bound FixL represents incomplete inhibition of kinase activity. Ultraviolet resonance Raman (UVRR) spectra revealed that oxygen binding to the truncated sensor domain of FixL markedly decreased the intensity of the Y8a band arising from Fα-10 Tyr. In contrast, no appreciable change in intensity of the Y8a band occurred after CO-binding, and time-resolved UVRR spectra of the sensor domain of FixL upon O2 dissociation indicated that structural changes near Fα-10 Tyr occurred at ~0.1 µs. These results suggest that oxygen binding to FixL changes protein conformation near the Fα-10 Tyr residue within a microsecond. The conformational changes of FixL upon O2 dissociation and underlying sensing mechanism also are discussed.
    The Journal of Physical Chemistry B 08/2013; 117(49). DOI:10.1021/jp406709e · 3.30 Impact Factor
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    ABSTRACT: Resonance Raman spectra of ligand-bound complexes including the 4-phenylimidazole complex and of free and L-Trp-bound forms of indoleamine 2, 3-dioxygenase in the ferric state were examined. Effects on the vinyl and propionate substituent groups of the heme were detected in a ligand-dependent fashion. The effects of phenyl group of 4-phenylimidazole on the vinyl and propionate Raman bands were evident when compared with the case of imidazole ligand. Substrate binding to the ferrous protein caused an upshift of the iron–histidine stretching mode by 3 cm−1, indicating an increase in negativity of the imidazole ring, which favors the O–O bond cleavage. The substrate binding event is likely to be communicated from the heme distal side to the iron–histidine bond through heme substituent groups and the hydrogen-bond network which includes water molecules, as identified in an X-ray structure of a 4-phenylimidazole complex. The results provide evidence for fine-tuning of the reactivity of O–O bond cleavage by the oxygenated heme upon binding of L-Trp.
    Chemical Physics 06/2013; 419:178–183. DOI:10.1016/j.chemphys.2013.02.032 · 1.65 Impact Factor
  • Takehiko Tosha · Yoshitsugu Shiro ·
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    ABSTRACT: Respiration is an essential biological process to get bioenergy, ATP, for all kingdoms of life. Cytochrome c oxidase (COX) plays central role in aerobic respiration, catalyzing the reduction of O(2) coupled with pumping proton across the biological membrane. Nitric oxide reductase (NOR) involved in anaerobic nitrate respiration is suggested to be evolutionary related to COX and share the same progenitor with COX, on the basis of the amino acid sequence homology. Contrary to COX, NOR catalyzes the reduction of nitric oxide and shows no proton pumping ability. Thus, the respiratory enzyme acquires (or loses) proton pumping ability in addition to the conversion of the catalytic property along with the environmental change on earth. Recently, we solved the structures of two types of NORs, which provides novel insights into the functional conversion of the respiratory enzymes. In this review, we focus on the structural similarities and differences between COXs and NORs and discuss possible mechanism for the functional conversion of these enzymes during molecular evolution. © 2013 IUBMB Life, 2013.
    International Union of Biochemistry and Molecular Biology Life 03/2013; 65(3). DOI:10.1002/iub.1135 · 3.14 Impact Factor

  • 01/2013; 51(10):679-685. DOI:10.1271/kagakutoseibutsu.51.679
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    ABSTRACT: Central CC coupling: It has been proposed that the heme-containing enzyme StaD and its homologues catalyze a coupling reaction that yields an indolepyruvic acid (IPA) imine in indolocarbazole biosynthesis. We show that StaD uses IPA enol as an analogue of natural substrate to produce an IPA dimer, in the first direct evidence of the CC coupling dimerization reaction catalyzed by StaD.
    ChemBioChem 11/2012; 13(17). DOI:10.1002/cbic.201200535 · 3.09 Impact Factor
  • Ryu Makino · Shinsuke Yazawa · Hiroshi Hori · Yoshitsugu Shiro ·
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    ABSTRACT: Nitric oxide (NO) elicits a wide variety of physiological responses by binding to the heme in soluble guanylate cyclase (sGC) to stimulate cGMP production. Although nucleotides, such as ATP or GTP analogs, have been reported to regulate the signaling of NO binding from the heme site to the catalytic site, the other regulatory functions of nucleotides remain unexamined. Among the nucleotides tested, we found that 2'-d-3'-GMP acted as a potent noncompetitive inhibitor with respect to Mn-GTP, when the ferrous enzyme combined with NO, CO or an allosteric activator BAY 41-2272. 2'-d-3'-GMP also displayed nearly identical patterns of inhibition for the ferric enzyme, in which the binding of N3- or BAY 41-2272 significantly increased the inhibitory effects of the nucleotide. Equilibrium dialysis measurements using the CO-ligated enzyme in the presence of allosteric activators demonstrated that 2'-d-3'-GMP exclusively binds to the catalytic site of sGC. Furthermore, the affinity of 2'-d-3'-GMP for the enzyme was found to increase upon addition of foscarnet, an analog of PPi. These findings together with other kinetic results imply that 2'-d-3'-GMP acts as a P-site inhibitor probably by forming a dead-end complex, sGC-2'-d-3'-GMP-PPi in the catalytic reaction. The complex formation of the enzyme with 2'-d-3'-GMP does not seem to be associated with changes in the Fe-proximal His bond strength, because the CO coordination state or the redox potentials of the enzyme-heme are virtually unaffected.
    Biochemistry 10/2012; 51(46). DOI:10.1021/bi3004044 · 3.02 Impact Factor
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    Y. Shiro ·

    Biochimica et Biophysica Acta (BBA) - Bioenergetics 10/2012; 1817:S103. DOI:10.1016/j.bbabio.2012.06.278 · 5.35 Impact Factor

Publication Stats

5k Citations
910.85 Total Impact Points


  • 2014
    • Hokkaido University
      • Faculty of Pharmaceutical Sciences
      Sapporo, Hokkaidō, Japan
  • 2003-2014
    • RIKEN
      • Biometal Science Laboratory
      Вако, Saitama, Japan
    • Brandeis University
      Waltham, Massachusetts, United States
    • University of Tsukuba
      • Institute of Applied Biochemistry
      Tsukuba, Ibaraki, Japan
  • 2010
    • The University of Tokushima
      • Department of Life Systems
      Tokusima, Tokushima, Japan
  • 2007
    • Nagoya University
      • Research Center for Materials Science
      Nagoya, Aichi, Japan
  • 2004
    • Toyota Physical and Chemical Institute
      Seto, Aichi, Japan
  • 2003-2004
    • Himeji Institute of Technology
      • Faculty of Science
      Himezi, Hyōgo, Japan
  • 2001
    • Keio University
      • School of Medicine
      Edo, Tokyo, Japan
  • 2000
    • University of Illinois, Urbana-Champaign
      • Beckman Institute for Advanced Science and Technology
      Urbana, Illinois, United States
  • 1979-1995
    • Kyoto University
      • • Department of Molecular Engineering
      • • Department of Energy and Hydrocarbon Chemistry
      Kioto, Kyōto, Japan