Nobuhiro Mizuno

University of Hyogo, Kōbe, Hyōgo, Japan

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Publications (10)55 Total impact

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    ABSTRACT: The features of hydrogen bonds in DsrD protein from sulfate-reducing bacteria have been investigated by neutron protein crystallography. The function of DsrD has not yet been elucidated clearly, but its X-ray crystal structure revealed that it comprises a winged-helix motif and shows the highest structural homology to the DNA-binding proteins. Since any neutron structure of a DNA recognition protein has not yet been obtained, here detailed information on the hydrogen bonds in the winged-helix-motif protein is given and the following features found. (i) The number of hydrogen bonds per amino acid of DsrD is relatively fewer than for other proteins for which neutron structures were determined previously. (ii) Hydrogen bonds are localized between main-chain and main-chain atoms; there are few hydrogen bonds between main-chain and side-chain atoms and between side-chain and side-chain atoms. (iii) Hydrogen bonds inducted by protonation of specific amino acid residues (Glu50) seem to play an essential role in the dimerization of DsrD. The former two points are related to the function of the DNA-binding protein; the three-dimensional structure was mainly constructed by hydrogen bonds in main chains, while the side chains appeared to be used for another role. The latter point would be expected to contribute to the crystal growth of DsrD.
    Journal of Synchrotron Radiation 06/2008; 15(Pt 3):277-80. · 2.19 Impact Factor
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    ABSTRACT: Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of beta-catenin by glycogen synthase kinase 3beta. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 91.49, c = 84.92 A. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 A.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2007; 63(Pt 6):529-31. · 0.55 Impact Factor
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    ABSTRACT: 6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.
    Journal of Biological Chemistry 12/2005; 280(47):39644-52. · 4.65 Impact Factor
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    ABSTRACT: To investigate the structure-function relationship between 6-aminohexanoate-dimer hydrolase (EII) from Arthrobacter sp. and a cryptic protein (EII') which shows 88% sequence identity to EII, a hybrid protein (named Hyb-24) of EII and EII' was overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in MES buffer pH 6.5. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 96.37, c = 113.09 A. Diffraction data were collected from native and methylmercuric chloride derivative crystals to resolutions of 1.75 and 1.80 A, respectively.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2005; 61(Pt 10):928-30. · 0.55 Impact Factor
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    ABSTRACT: Neutron crystallography can provide a substantial amount of information about the hydration of proteins. The hydration patterns of three proteins, whose structures have been solved at 1.5 or 1.6 A resolution using our BIX-type diffractometers, show interesting features. The water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen-bond formation and dynamics of hydration. In addition, the neutron diffraction study of a DNA-binding protein, dissimilatory sulfite reductase D (DsrD) is briefly described, and some preliminary results are presented. This topic is of interest because it is well known that hydrogen bonds play important roles in DNA-protein recognition.
    Journal of Synchrotron Radiation 02/2004; 11(Pt 1):72-5. · 2.19 Impact Factor
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    ABSTRACT: Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for a neutron diffraction study. The initial crystals obtained were too small for the neutron experiment. In order to obtain a larger crystal (>1 mm3), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal-quality assessment via X-ray diffraction. Using conditions determined in this manner, a large single crystal (1.7 mm3) of DsrD protein was subsequently grown in D(2)O solution by the macroseeding technique. A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI), collecting data to 2.4 A resolution from an optimized crystal.
    Acta Crystallographica Section D Biological Crystallography 12/2003; 59(Pt 12):2306-9. · 14.10 Impact Factor
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    ABSTRACT: The crystal structure of DsrD from Desulfovibrio vulgaris Hildenborough has been determined at 1.2 A resolution. DsrD is in a dimeric form in the crystal, and five sulfate anions were located on the surface. The structure of DsrD comprises a winged-helix motif, which shows the highest structural homology to similar motifs found in Z-DNA binding proteins and some B-DNA binding proteins. The core structure of the molecule is constructed by intramolecular interactions of hydrophobic residues, which are well conserved in DNA binding proteins, suggesting that these proteins belong to the same superfamily on the basis of the structure. These results indicate a possible role of DsrD in transcription or translation of genes for enzymes catalyzing dissimilatory sulfite reduction.
    Structure 10/2003; 11(9):1133-40. · 5.99 Impact Factor
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    ABSTRACT: The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H(2) in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 A and 160 degrees, respectively, except for one case (1.72 A and 135 degrees ), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H(2)-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm ( approximately 3000 M(-1)cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (13)C(18)O). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography.
    Journal of the American Chemical Society 11/2002; 124(39):11628-35. · 10.68 Impact Factor
  • Acta Crystallographica Section A - ACTA CRYSTALLOGR A. 01/2002; 58.
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    ABSTRACT: DsrD (dissimilatory sulfite reductase D) protein encoded by the dsr operon of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has been crystallized using the vapour-diffusion method with ammonium sulfate as a precipitating agent. The crystals diffract to 1.7 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 60.54 (6), b = 65. 20 (4), c = 46.41 (3) A. The crystal contains two DsrD molecules per asymmetric unit, giving a Matthews coefficient (V(M)) of 2.6 A(3) Da(-1). A gold-derivative (NaAuCl(4)) crystal has been successfully prepared.
    Acta Crystallographica Section D Biological Crystallography 07/2000; 56(Pt 6):754-5. · 14.10 Impact Factor

Publication Stats

110 Citations
55.00 Total Impact Points


  • 2005–2008
    • University of Hyogo
      • • Department of Life Science
      • • Department of Materials Science and Chemistry (Graduate)
      Kōbe, Hyōgo, Japan
  • 2000–2005
    • Kyoto University
      • • Department of Cell and Developmental Biology
      • • Division of Chemistry
      Kioto, Kyōto, Japan