Muraki, Norifumi



Faculty of Science and Technology, Department of Chemistry (Yagami)


Associate Professor (Non-tenured)


Papers 【 Display / hide

  • Characterization of a novel cysteine-less Cu/Zn-superoxide dismutase in Paenibacillus lautus missing a conserved disulfide bond

    Furukawa Y., Shintani A., Narikiyo S., Sue K., Akutsu M., Muraki N.

    Journal of Biological Chemistry (Journal of Biological Chemistry)  299 ( 8 )  2023.08

    ISSN  00219258

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    Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.

  • Intrinsic structural vulnerability in the hydrophobic core induces species-specific aggregation of canine SOD1 with degenerative myelopathy–linked E40K mutation

    Hashimoto K., Watanabe S., Akutsu M., Muraki N., Kamishina H., Furukawa Y., Yamanaka K.

    Journal of Biological Chemistry (Journal of Biological Chemistry)  299 ( 6 )  2023.06

    ISSN  00219258

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    Canine degenerative myelopathy (DM), a fatal neurodegenerative disease in dogs, shares clinical and genetic features with amyotrophic lateral sclerosis, a human motor neuron disease. Mutations in the SOD1 gene encoding Cu/Zn superoxide dismutase (SOD1) cause canine DM and a subset of inherited human amyotrophic lateral sclerosis. The most frequent DM causative mutation is homozygous E40K mutation, which induces the aggregation of canine SOD1 but not of human SOD1. However, the mechanism through which canine E40K mutation induces species-specific aggregation of SOD1 remains unknown. By screening human/canine chimeric SOD1s, we identified that the humanized mutation of the 117th residue (M117L), encoded by exon 4, significantly reduced aggregation propensity of canine SOD1E40K. Conversely, introducing a mutation of leucine 117 to methionine, a residue homologous to canine, promoted E40K-dependent aggregation in human SOD1. M117L mutation improved protein stability and reduced cytotoxicity of canine SOD1E40K. Furthermore, crystal structural analysis of canine SOD1 proteins revealed that M117L increased the packing within the hydrophobic core of the β-barrel structure, contributing to the increased protein stability. Our findings indicate that the structural vulnerability derived intrinsically from Met 117 in the hydrophobic core of the β-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.

  • Crystal structural analysis of aldoxime dehydratase from Bacillus sp. OxB-1: Importance of surface residues in optimization for crystallization

    Matsui D., Muraki N., Chen K., Mori T., Ingram A.A., Oike K., Gröger H., Aono S., Asano Y.

    Journal of Inorganic Biochemistry (Journal of Inorganic Biochemistry)  230 2022.05

    ISSN  01620134

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    Aldoxime dehydratase (Oxd) is a heme enzyme that catalyzes aldoxime dehydration to the corresponding nitriles. Unlike many other heme enzymes, Oxd has a unique feature that the substrate binds directly to the heme. Therefore, it is thought that structural differences around the bound heme directly relate to differences in substrate selection. However sufficient structural information to discuss the substrate specificity has not been obtained. Oxd from Bacillus sp. OxB-1 (OxdB) shows unique substrate specificity and enantioselectivity compared to the Oxds whose crystal structures have already been reported. Here, we report the crystal structure of OxdB, which has not been reported previously. Although the crystallization of OxdB has been difficult, by adding a site-specific mutation to Glu85 located on the surface of the protein, we succeeded in crystallizing OxdB without reducing the enzyme activity. The catalytic triad essential for Oxd activity were structurally conserved in OxdB. In addition, the crystal structure of the Michaelis complex of OxdB and the diastereomerically pure substrate Z-2-(3-bromophenyl)-propanal oxime implied the importance of several hydrophobic residues for substrate specificity. Mutational analysis implicated Ala12 and Ala14 in the E/Z selectivity of bulky compounds. The N-terminal region of OxdB was shown to be shorter than those of Oxds from Pseudomonas chlororaphis and Rhodococcus sp. N-771, and have high flexibility. These structural differences possibly result in distinct preferences for aldoxime substrates based on factors such as substrate size.

  • Heme controls the structural rearrangement of its sensor protein mediating the hemolytic bacterial survival

    Nishinaga M., Sugimoto H., Nishitani Y., Nagai S., Nagatoishi S., Muraki N., Tosha T., Tsumoto K., Aono S., Shiro Y., Sawai H.

    Communications Biology (Communications Biology)  4 ( 1 )  2021.12

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    Hemes (iron-porphyrins) are critical for biological processes in all organisms. Hemolytic bacteria survive by acquiring b-type heme from hemoglobin in red blood cells from their animal hosts. These bacteria avoid the cytotoxicity of excess heme during hemolysis by expressing heme-responsive sensor proteins that act as transcriptional factors to regulate the heme efflux system in response to the cellular heme concentration. Here, the underlying regulatory mechanisms were investigated using crystallographic, spectroscopic, and biochemical studies to understand the structural basis of the heme-responsive sensor protein PefR from Streptococcus agalactiae, a causative agent of neonatal life-threatening infections. Structural comparison of heme-free PefR, its complex with a target DNA, and heme-bound PefR revealed that unique heme coordination controls a >20 Å structural rearrangement of the DNA binding domains to dissociate PefR from the target DNA. We also found heme-bound PefR stably binds exogenous ligands, including carbon monoxide, a by-product of the heme degradation reaction.

  • Structural characterization of y29f mutant of thermoglobin from a hyperthermophilic bacterium aquifex aeolicus

    Muraki N., Takeda K., Nam D., Muraki M., Aono S.

    Chemistry Letters (Chemistry Letters)  50 ( 4 )  2021

    ISSN  03667022

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    We have determined the crystal structure of thermoglobin (AaTgb) from a hyperthermophilic bacterium Aquifex aeolicus. Tyrosine and glutamine at the B10 and E7 position, respectively, are conserved in AaTgb as are the case of single domain hemoglobins (sdHbs). While the binding affinity of O2 or CO is affected by the replacement of Tyr29 in the distal heme pocket, wild type and Y29F variant of AaTgb show a similar binding affinity of imidazole.

Research Projects of Competitive Funds, etc. 【 Display / hide

  • 金属酵素の成熟化に関わる錯体生合成複合体の構造機能解明


    基盤研究(C), Principal investigator

  • NiFe型ヒドロゲナーゼの成熟化における一酸化炭素の輸送機構の解明


    MEXT,JSPS, Grant-in-Aid for Scientific Research, 基盤研究(C), Principal investigator


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