CVClient

柴田 直樹

シバタ ナオキ  (Naoki Shibata)

基本情報

所属
兵庫県立大学 大学院理学研究科生命科学専攻 助教授・准教授
学位
博士(工学)(大阪大学)

J-GLOBAL ID
200901039667162990
researchmap会員ID
1000224235

外部リンク

論文

 138
  • Seiji Negoro, Naoki Shibata, Dai-Ichiro Kato, Yusuke Tanaka, Kengo Yasuhira, Keisuke Nagai, Shohei Oshima, Yoko Furuno, Risa Yokoyama, Kaito Miyazaki, Masahiro Takeo, Kowit Hengphasatporn, Yasuteru Shigeta, Young-Ho Lee, Yoshiki Higuchi
    The FEBS journal 2023年6月5日  査読有り
    Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10-5  s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.
  • Naoki Shibata, Tetsuo Toraya
    Chembiochem : a European journal of chemical biology e202300021 2023年3月14日  査読有り招待有り筆頭著者責任著者
    Adenosylcobalamin (AdoCbl) or coenzyme B12 is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes and generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co-C) bond for catalysis. How do the enzymes activate and cleave the Co-C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase established the crucial importance of steric strain of the Co-C bond and conformational stabilization of adenosyl radical for coenzyme B12 catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B12 enzymes.
  • Naoki Shibata, Yoshiki Higuchi, Bernhard Kräutler, Tetsuo Toraya
    Chemistry (Weinheim an der Bergstrasse, Germany) 28(65) e202202196 2022年11月21日  査読有り筆頭著者責任著者
    The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.
  • Tetsuo Toraya, Takamasa Tobimatsu, Koichi Mori, Mamoru Yamanishi, Naoki Shibata
    Methods in enzymology 668 181-242 2022年  査読有り招待有り最終著者
    Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.
  • Tetsuo Toraya, Takamasa Tobimatsu, Naoki Shibata, Koichi Mori
    Methods in enzymology 668 243-284 2022年  査読有り招待有り
    Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

MISC

 65
  • 柴田直樹, 樋口芳樹, 虎谷哲夫
    第468回ビタミンB研究協議会 2022年9月  筆頭著者
  • 根来誠司, 武尾正弘, 柴田直樹, 樋口芳樹, 加藤太一郎, 重田育照
    月刊バイオインダストリー 2019年6月  招待有り
  • Yamanaka Masaru, Hoshizumi Makoto, Nagao Satoshi, Nakayama Ryoko, Shibata Naoki, Higuchi Yoshiki, Hirota Shun
    2017年2月14日  
    The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein.
  • 衣笠 凌, 羽田 圭吾, 谷本 悠樹, 竹原 一起, 柴田 直樹, 加藤 太一郎, 武尾 正弘, 根来 誠司
    日本生物工学会大会講演要旨集 67 282-282 2015年  
  • Y. Higuchi, M. Akter, C. Inoue, Y. Shomura, N. Shibata, K. Inaka, K. Kataoka, T. Sakurai, K. Komori
    JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 19 S324-S324 2014年3月  

書籍等出版物

 3

主要な共同研究・競争的資金等の研究課題

 20