Takeshi Hayashi

Beppu University, Бэппу, Ōita, Japan

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Publications (7)29.1 Total impact

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    ABSTRACT: SCO4008 from Streptomyces coelicolor A3(2) is a member of the TetR family. However, its precise function is not yet clear. In this study, the crystal structure of SCO4008 was determined at a resolution of 2.3 Å, and its DNA-binding properties were analyzed. Crystal structure analysis showed that SCO4008 forms an -shaped homodimer, in which the monomer is composed of an N-terminal DNA-binding domain (N-DBD) containing a helix-turn-helix (HTH) and C-terminal dimerization and regulatory domain (C-DRD) possessing a ligand-binding cavity. The genomic systematic evolution of ligands by exponential enrichment (genomic SELEX) and electrophoretic mobility shift assay (EMSA) revealed that four SCO4008 dimers bind to the two operator regions located between sco4008 and sco4007, a secondary transporter belonging to the major facilitator superfamily. Ligand screening analysis showed that SCO4008 recognizes a wide range of structurally dissimilar cationic and hydrophobic compounds. These results suggested that SCO4008 is a transcriptional repressor of sco4007 responsible for the multidrug resistance system in S. coelicolor A3(2).
    Journal of Molecular Biology 07/2013; · 3.91 Impact Factor
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    ABSTRACT: SCO5550 from the model actinomycete Streptomyces coelicolor A3(2) was identified as a putative transcriptional regulator, and classified into the MerR family by sequence analysis. Recombined SCO5550 was successfully produced in Rhodococcus erythropolis, which can be used to stably express recombinant protein by optimizing the temperature over a wide range (4°C - 35°C). Crystal structure analysis showed that the dimerization domain (C-terminal domain) of SCO5550 has a novel fold and forms a new dimer shape, whereas the DNA-binding domain (N-terminal domain) is very similar to those of MerR family members. Such the new dimer form suggests that SCO5550 may define a new subfamily as a new member of the MerR family. Binding DNA sequence analysis of SCO5550 using the genomic systematic evolution of ligands by exponential enrichment (gSELEX) and electrophoretic mobility shift assay (EMSA) indicated that SCO5550 regulates the expression of the immediately upstream gene sco5551 encoding a putative protein, probably as a transcriptional activator.
    Biochemical and Biophysical Research Communications 04/2013; · 2.41 Impact Factor
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    Takeshi Hayashi, Tsuyoshi Kato, Kensuke Furukawa
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    ABSTRACT: We previously isolated respiratory-deficient mutant (RDM) strains of Zymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochrome bd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within the ndh gene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.
    Applied and environmental microbiology 06/2012; 78(16):5622-9. · 3.69 Impact Factor
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    ABSTRACT: We encountered a fourth case of honey allergy in Japan. We characterized and identified the IgE-binding proteins in honey using the serum of a honey-allergenic patient. Immunoblot analysis revealed that IgE in the patient serum specifically bound to four proteins in each honey sample. At least three of these IgE-binding proteins were N-linked glycoproteins. To identify the 60-kDa IgE-binding protein in dandelion honey, the N-terminal sequences of the fragmented protein were analyzed, revealing the protein to be major royal jelly protein 1 (MRJP 1). Three IgE-binding proteins removed of N-linked oligosaccharide showed a large reduction in IgE-binding activity as compared with the intact protein. This suggests that the carbohydrates in the IgE-binding proteins are a major epitope for patient IgE.
    Bioscience Biotechnology and Biochemistry 03/2011; 75(3):556-60. · 1.27 Impact Factor
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    ABSTRACT: Respiration-deficient mutant (RDM) strains of Zymomonas mobilis were isolated from antibiotic-resistant mutants. These RDM strains showed various degrees of respiratory deficiency. All RDM strains exhibited much higher ethanol fermentation capacity than the wild-type strain under aerobic conditions. The strains also gained thermotolerance and exhibited greater ethanol production at high temperature (39°C), under both non-aerobic and aerobic conditions, compared with the wild-type strain. Microarray and subsequent quantitative PCR analyses suggest that enhanced gene expression involved in the metabolism of glucose to ethanol resulted in the high ethanol production of RDM strains under aerobic growth conditions. Reduction of intracellular oxidative stress may also result in improved ethanol fermentation by RDM strains at high temperatures.
    Journal of Bioscience and Bioengineering 01/2011; 111(4):414-9. · 1.74 Impact Factor
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    ABSTRACT: SCO0332 protein is a putative TetR-family transcriptional regulator from Streptomyces coelicolor A3(2). The crystal structure of SCO0332 was determined at 2.25 A resolution by single-wavelength anomalous diffraction (SAD) phasing using the S atoms of the native protein. SCO0332 contains a helix-turn-helix (HTH) DNA-binding motif in its N-terminal region and forms a homodimer. The overall structure of SCO0332 shows significant similarity to other TetR-family regulators. A systematic evolution of ligands by exponential enrichment (SELEX) analysis indicated that SCO0332 has sequence-specific DNA-binding ability and determined the position of the operator element of SCO0332 on the chromosomal DNA of S. coelicolor. An electrophoretic mobility-shift assay (EMSA) showed that SCO0332 binds to the operator sequence upstream of the sco0330 gene, which encodes a putative short-chain oxidoreductase. These results suggest that SCO0332 is a transcriptional repressor that regulates sco0330 gene expression.
    Acta Crystallographica Section D Biological Crystallography 03/2008; 64(Pt 2):198-205. · 14.10 Impact Factor
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    ABSTRACT: SCO6571 protein from Streptomyces coelicolor A3(2) was overexpressed and purified using Rhodococcus erythropolis as an expressing host. Crystals of selenomethionine-substituted SCO6571 have been obtained by vapor diffusion method. SCO6571 crystals diffract to 2.3 A and were found to belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters a = 84.5, b = 171.6, c = 184.8 A. Six molecules in the asymmetric unit give a crystal volume per protein mass (V(M)) of 2.97 A (3) Da(-1) and solvent content of 58.6 %. The structure was solved by the single wavelength anomalous diffraction (SAD) method. SCO6571 is a TIM-barrel fold protein that assembles into a hexameric molecule with D(3) symmetry.
    Protein and Peptide Letters 02/2008; 15(7):709-12. · 1.99 Impact Factor