Yoshitsugu Shiro

Hokkaido University, Sapporo, Hokkaidō, Japan

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Publications (218)937.57 Total impact

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    ACS Catalysis 01/2015; 5:150-159. DOI:10.1021/cs501592f · 7.57 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; DOI:10.1074/jbc.M114.598391 · 4.60 Impact Factor
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e5. DOI:10.1016/j.bbabio.2014.05.125 · 4.83 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 · 2.92 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 · 11.34 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 · 3.27 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 · 6.72 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 · 4.79 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 · 3.38 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 · 2.03 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 · 2.76 Impact Factor
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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 · 3.06 Impact Factor
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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 · 3.19 Impact Factor
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    ABSTRACT: The stereoselective epoxidation of styrene was catalyzed by H(2) O(2) -dependent cytochrome P450(SPα) in the presence of carboxylic acids as decoy molecules. The stereoselectivity of styrene oxide could be altered by the nature of the decoy molecules. In particular, the chirality at the α-positions of the decoy molecules induced a clear difference in the chirality of the product: (R)-ibuprofen enhanced the formation of (S)-styrene oxide, whereas (S)-ibuprofen preferentially afforded (R)-styrene oxide. The crystal structure of an (R)-ibuprofen-bound cytochrome P450(SPα) (resolution 1.9 Å) revealed that the carboxylate group of (R)-ibuprofen served as an acid-base catalyst to initiate the epoxidation. A docking simulation of the binding of styrene in the active site of the (R)-ibuprofen-bound form suggested that the orientation of the vinyl group of styrene in the active site agreed with the formation of (S)-styrene oxide.
    Chemistry - An Asian Journal 10/2012; 7(10):2286-93. DOI:10.1002/asia.201200250 · 3.94 Impact Factor
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    ABSTRACT: Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666-70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb(3) oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.
    PLoS Computational Biology 08/2012; 8(8):e1002674. DOI:10.1371/journal.pcbi.1002674 · 4.83 Impact Factor
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    ABSTRACT: Although heme is a crucial element for many biological processes including respiration, heme homeostasis should be regulated strictly due to the cytotoxicity of free heme molecules. Numerous lactic acid bacteria, including Lactococcus lactis, acquire heme molecules exogenously to establish an aerobic respiratory chain. A heme efflux system plays an important role for heme homeostasis to avoid cytotoxicity of acquired free heme, but its regulatory mechanism is not clear. Here, we report that the transcriptional regulator heme-regulated transporter regulator (HrtR) senses and binds a heme molecule as its physiological effector to regulate the expression of the heme-efflux system responsible for heme homeostasis in L. lactis. To elucidate the molecular mechanisms of how HrtR senses a heme molecule and regulates gene expression for the heme efflux system, we determined the crystal structures of the apo-HrtR·DNA complex, apo-HrtR, and holo-HrtR at a resolution of 2.0, 3.1, and 1.9 Å, respectively. These structures revealed that HrtR is a member of the TetR family of transcriptional regulators. The residue pair Arg-46 and Tyr-50 plays a crucial role for specific DNA binding through hydrogen bonding and a CH-π interaction with the DNA bases. HrtR adopts a unique mechanism for its functional regulation upon heme sensing. Heme binding to HrtR causes a coil-to-helix transition of the α4 helix in the heme-sensing domain, which triggers a structural change of HrtR, causing it to dissociate from the target DNA for derepression of the genes encoding the heme efflux system. HrtR uses a unique heme-sensing motif with bis-His (His-72 and His-149) ligation to the heme, which is essential for the coil-to-helix transition of the α4 helix upon heme sensing.
    Journal of Biological Chemistry 07/2012; 287(36):30755-68. DOI:10.1074/jbc.M112.370916 · 4.60 Impact Factor
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    ABSTRACT: The crystal structure of a truncated Aer2, a signal transducer protein from Pseudomonas aeruginosa, consisting of the heme-containing PAS and di-HAMP domains revealed that a distal tryptophan residue (Trp283) plays an important role in stabilizing the heme-bound O(2) and intra-molecular signal transduction upon O(2) binding.
    Chemical Communications 05/2012; 48(52):6523-5. DOI:10.1039/c2cc32549g · 6.72 Impact Factor

Publication Stats

4k Citations
937.57 Total Impact Points


  • 2014
    • Hokkaido University
      Sapporo, Hokkaidō, Japan
  • 1999–2014
    • RIKEN
      • Biometal Science Laboratory
      Вако, Saitama, Japan
  • 2009–2012
    • SPring-8
      Saitama, Saitama, Japan
  • 2007–2012
    • Nagoya University
      • • Department of Chemistry
      • • Research Center for Materials Science
      Nagoya, Aichi, Japan
  • 2003–2007
    • Himeji Institute of Technology
      • • Graduate School of Science
      • • Faculty of Science
      Himezi, Hyōgo, Japan
    • Brandeis University
      Waltham, Massachusetts, United States
  • 2006
    • University of Hyogo
      • Department of Life Science
      Kōbe, Hyōgo, Japan
  • 1995–2005
    • Gakushuin University
      • Faculty of Science
      Edo, Tōkyō, Japan
  • 2004
    • Yamagata University
      Ямагата, Yamagata, Japan
    • Toyota Physical and Chemical Institute
      Seto, Aichi, Japan
  • 1994–2003
    • University of Tsukuba
      • Institute of Applied Biochemistry
      Tsukuba, Ibaraki, Japan
  • 2002
    • Osaka City University
      • Graduate School of Medicine
      Ōsaka-shi, Osaka-fu, Japan
    • Keio University
      • Department of Physics
      Edo, Tōkyō, Japan
    • Hosei University
      Edo, Tōkyō, Japan
  • 1979–2002
    • Kyoto University
      • • Department of Molecular Engineering
      • • Department of Energy and Hydrocarbon Chemistry
      Kioto, Kyōto, Japan
  • 2000
    • Kyoritsu College of Pharmacy
      Edo, Tōkyō, Japan
    • University of Illinois, Urbana-Champaign
      • Beckman Institute for Advanced Science and Technology
      Urbana, Illinois, United States
  • 1995–1997
    • Chuo University
      • Department of Applied Chemistry
      Edo, Tōkyō, Japan