研究者詳細 - 加藤　公児

Structural basis for tRNA-dependent cysteine biosynthesis 査読

Meirong Chen, Koji Kato, Yume Kubo, Yoshikazu Tanaka, Yuchen Liu, Feng Long, William B. Whitman, Pascal Lill, Christos Gatsogiannis, Stefan Raunser, Nobutaka Shimizu, Akira Shinoda, Akiyoshi Nakamura, Isao Tanaka, Min Yao

NATURE COMMUNICATIONS 8 ( 1 ) 1521 2017年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Cysteine can be synthesized by tRNA-dependent mechanism using a two-step indirect pathway, where O-phosphoseryl-tRNA synthetase (SepRS) catalyzes the ligation of a mismatching O-phosphoserine (Sep) to tRNA(Cys) followed by the conversion of tRNA-bounded Sep into cysteine by Sep-tRNA:Cys-tRNA synthase (SepCysS). In ancestral methanogens, a third protein SepCysE forms a bridge between the two enzymes to create a ternary complex named the transsulfursome. By combination of X-ray crystallography, SAXS and EM, together with biochemical evidences, here we show that the three domains of SepCysE each bind SepRS, SepCysS, and tRNA(Cys), respectively, which mediates the dynamic architecture of the transsulfursome and thus enables a global long-range channeling of tRNA(Cys) between SepRS and SepCysS distant active sites. This channeling mechanism could facilitate the consecutive reactions of the two-step indirect pathway of Cys-tRNA(Cys) synthesis (tRNA-dependent cysteine biosynthesis) to prevent challenge of translational fidelity, and may reflect the mechanism that cysteine was originally added into genetic code.

DOI： 10.1038/s41467-017-01543-y

A novel glycoside hydrolase family 97 enzyme: Bifunctional β-l-arabinopyranosidase/α-galactosidase from Bacteroides thetaiotaomicron. 査読

Kikuchi A, Okuyama M, Kato K, Osaki S, Ma M, Kumagai Y, Matsunaga K, Klahan P, Tagami T, Yao M, Kimura A

Biochimie 142 41 - 50 2017年11月

Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C 査読

Shingo Nojima, Ayumi Fujishima, Koji Kato, Kayoko Ohuchi, Nobutaka Shimizu, Kento Yonezawa, Kenji Tajima, Min Yao

SCIENTIFIC REPORTS 7 ( 1 ) 13018 2017年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Bacterial cellulose (BC) is synthesized and exported through the cell membrane via a large protein complex (terminal complex) that consists of three or four subunits. BcsC is a little-studied subunit considered to export BC to the extracellular matrix. It is predicted to have two domains: a tetratrico peptide repeat (TPR) domain and a beta-barrelled outer membrane domain. Here we report the crystal structure of the N-terminal part of BcsC-TPR domain (Asp24-Arg272) derived from Enterobacter CJF-002. Unlike most TPR-containing proteins which have continuous TPR motifs, this structure has an extra a-helix between two clusters of TPR motifs. Five independent molecules in the crystal had three different conformations that varied at the hinge of the inserted a-helix. Such structural feature indicates that the inserted a-helix confers flexibility to the chain and changes the direction of the TPR super-helix, which was also suggested by structural analysis of BcsC-TPR (Asp24-Leu664) in solution by size exclusion chromatography-small-angle X-ray scattering. The flexibility at the a-helical hinge may play important role for exporting glucan chains.

DOI： 10.1038/s41598-017-12530-0

Crystallographic analysis of a subcomplex of the transsulfursome with tRNA for Cys-tRNA(Cys) synthesis 査読

Meirong Chen, Yuto Nakazawa, Yume Kubo, Nozomi Asano, Koji Kato, Isao Tanaka, Min Yao

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 72 ( Pt 7 ) 569 - 572 2016年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INT UNION CRYSTALLOGRAPHY

In most organisms, Cys-tRNA(Cys) is directly synthesized by cysteinyl-tRNA synthetase (CysRS). Many methanogenic archaea, however, use a two-step, indirect pathway to synthesize Cys-tRNA(Cys) owing to a lack of CysRS and cysteine-biosynthesis systems. This reaction is catalyzed by O-phosphoseryl-tRNA synthetase (SepRS), Sep-tRNA:Cys-tRNA synthase (SepCysS) and SepRS/ SepCysS pathway enhancer (SepCysE) as the transsulfursome, in which SepCysE connects both SepRS and SepCysS. On the transsulfursome, SepRS first ligates an O-phosphoserine to tRNA(Cys), and the mischarged intermediate Sep-tRNA(Cys) is then transferred to SepCysS, where it is further modified to Cys-tRNA(Cys). In this study, a subcomplex of the transsulfursome with tRNA(Cys) (SepCysS-SepCysE-tRNA(Cys)), which is involved in the second reaction step of the indirect pathway, was constructed and then crystallized. The crystals diffracted X-rays to a resolution of 2.6 angstrom and belonged to space group P6(5)22, with unit-cell parameters a = b = 107.2, c = 551.1 angstrom. The structure determined by molecular replacement showed that the complex consists of a SepCysS dimer, a SepCysE dimer and one tRNA(Cys) in the asymmetric unit.

DOI： 10.1107/S2053230X16009559

Template-dependent nucleotide addition in the reverse (3'-5') direction by Thg1-like protein 査読

Shoko Kimura, Tateki Suzuki, Meirong Chen, Koji Kato, Jian Yu, Akiyoshi Nakamura, Isao Tanaka, Min Yao

SCIENCE ADVANCES 2 ( 3 ) e1501397 2016年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC ADVANCEMENT SCIENCE

Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5'-end of truncated transfer RNA(tRNA) species in a Watson-Crick template-dependent manner. The reaction proceeds in two steps: the activation of the 5'-end by adenosine 5'-triphosphate (ATP)/guanosine 5'-triphosphate (GTP), followed by nucleotide addition. Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3'-5' direction. The enzyme creates two substrate binding sites for the first-and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3'-OH of the incoming nucleotide and the 5'-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. That the 3'-5' elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNA(His)-specific G(-1) addition enzyme. Each tRNA(His) binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible beta-hairpin. Furthermore, mutational analyses show that tRNA(His) is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode.

DOI： 10.1126/sciadv.1501397

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies 査読

Yuxin Ye, Wataru Saburi, Rei Odaka, Koji Kato, Naofumi Sakurai, Keisuke Komoda, Mamoru Nishimoto, Motomitsu Kitaoka, Haruhide Mori, Min Yao

FEBS LETTERS 590 ( 6 ) 828 - 837 2016年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

In Ruminococcus albus, 4-Omicron-beta-D-mannosyl-D-glucose phosphorylase (RaMP1) and beta-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze beta-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-Omicron-beta-D-mannosyl-D-glucose and RaMP2 with/without beta-(1 -> 4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.

DOI： 10.1002/1873-3468.12105

Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 Å Resolution. 査読

Gai Z, Matsuno A, Kato K, Kato S, Khan MR, Shimizu T, Yoshioka T, Kato Y, Kishimura H, Kanno G, Miyabe Y, Terada T, Tanaka Y, Yao M

Structure (London, England : 1993) 23 ( 12 ) 2204 - 2212 2015年12月

Structural basis for pore-forming mechanism of staphylococcal α-hemolysin. 査読

Sugawara T, Yamashita D, Kato K, Peng Z, Ueda J, Kaneko J, Kamio Y, Tanaka Y, Yao M

Toxicon : official journal of the International Society on Toxinology 108 226 - 231 2015年12月

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism. 査読

Shen X, Saburi W, Gai Z, Kato K, Ojima-Kato T, Yu J, Komoda K, Kido Y, Matsui H, Mori H, Yao M

Acta crystallographica. Section D, Biological crystallography 71 ( Pt 6 ) 1382 - 1391 2015年6月

Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin 査読

Asuka Matsuno, Zuoqi Gai, Miyuki Tanaka, Koji Kato, Sanae Kato, Tsuyoshi Katoh, Takeshi Shimizu, Takeya Yoshioka, Hideki Kishimura, Yoshikazu Tanaka, Min Yao

JOURNAL OF STRUCTURAL BIOLOGY 190 ( 3 ) 379 - 382 2015年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Many molluscs transport oxygen using a very large cylindrical multimeric copper-containing protein named hemocyanin. The molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330-450 kDa subunits, resulting in a molecular mass >3.3 MDa. Therefore, molluscan hemocyanin is one of the largest proteins. The reason why these organisms use such a large supermolecule for oxygen transport remains unclear. Atomic-resolution X-ray crystallographic analysis is necessary to unveil the detailed molecular structure of this mysterious large molecule. However, its propensity to dissociate in solution has hampered the crystallization of its intact form. In the present study, we successfully obtained the first crystals of an intact decameric molluscan hemocyanin. The diffraction dataset at 3.0-angstrom resolution was collected by merging the datasets of two isomorphic crystals. Electron microscopy analysis of the dissolved crystals revealed cylindrical particles. Furthermore, self-rotation function analysis clearly showed the presence of a fivefold symmetry with several twofold symmetries perpendicular to the fivefold axis. The absorption spectrum of the crystals showed an absorption peak around 345 nm. These results indicated that the crystals contain intact hemocyanin decamers in the oxygen-bound form. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jsb.2015.04.015

Structural and functional analysis of the Rpf2-Rrs1 complex in ribosome biogenesis 査読

Nozomi Asano, Koji Kato, Akiyoshi Nakamura, Keisuke Komoda, Isao Tanaka, Min Yao

NUCLEIC ACIDS RESEARCH 43 ( 9 ) 4746 - 4757 2015年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

Proteins Rpf2 and Rrs1 are required for 60S ribosomal subunit maturation. These proteins are necessary for the recruitment of three ribosomal components (5S ribosomal RNA [rRNA], RpL5 and RpL11) to the 90S ribosome precursor and subsequent 27SB pre-rRNA processing. Here we present the crystal structure of the Aspergillus nidulans (An) Rpf2-Rrs1 core complex. The core complex contains the tightly interlocked N-terminal domains of Rpf2 and Rrs1. The Rpf2 N-terminal domain includes a Brix domain characterized by similar N-and C-terminal architecture. The long alpha-helix of Rrs1 joins the C-terminal half of the Brix domain as if it were part of a single molecule. The conserved proline-rich linker connecting the N- and C-terminal domains of Rrs1 wrap around the side of Rpf2 and anchor the C-terminal domain of Rrs1 to a specific site on Rpf2. In addition, gel shift analysis revealed that the Rpf2-Rrs1 complex binds directly to 5S rRNA. Further analysis of Rpf2-Rrs1 mutants demonstrated that Saccha-romyces cerevisiae Rpf2 R236 (corresponds to R238 of AnRpf2) plays a significant role in this binding. Based on these studies and previous reports, we have proposed a model for ribosomal component recruitment to the 90S ribosome precursor.

DOI： 10.1093/nar/gkv305

Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate-binding site in GH13 dextran glucosidase 査読

Momoko Kobayashi, Wataru Saburi, Daichi Nakatsuka, Hironori Hondoh, Koji Kato, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Min Yao

FEBS LETTERS 589 ( 4 ) 484 - 489 2015年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Streptococcus mutans dextran glucosidase (SmDG) belongs to glycoside hydrolase family 13, and catalyzes both the hydrolysis of substrates such as isomaltooligosaccharides and subsequent transglucosylation to form alpha-(1 -> 6)-glucosidic linkage at the substrate non-reducing ends. Here, we report the 2.4 angstrom resolution crystal structure of glucosyl-enzyme intermediate of SmDG. In the obtained structure, the Trp238 side-chain that constitutes the substrate-binding site turned away from the active pocket, concurrently with conformational changes of the nucleophile and the acid/base residues. Different conformations of Trp238 in each reaction stage indicated its flexibility. Considering the results of kinetic analyses, such flexibility may reflect a requirement for the reaction mechanism of SmDG. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2015.01.005

Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis 査読

Tateki Suzuki, Akiyoshi Nakamura, Koji Kato, Dieter Soell, Isao Tanaka, Kelly Sheppard, Min Yao

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 112 ( 2 ) 382 - 387 2015年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNAAsn may assemble in an similar to 400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA(Asn). We report the 3.7-angstrom resolution crystal structure of the Pseudomonas aeruginosa A-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA(Asn) molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNA(Asn) formation evolutionarily preceded the direct attachment of Asn to tRNA(Asn), our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

DOI： 10.1073/pnas.1423314112

Crystallization and preliminary X-ray crystallographic analysis of ribosome assembly factors: The Rpf2-Rrs1 complex 査読

Nozomi Asano, Akiyoshi Nakamura, Keisuke Komoda, Koji Kato, Isao Tanaka, Min Yao

Acta Crystallographica Section F:Structural Biology Communications 70 1649 - 1652 2014年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Union of Crystallography

Rpf2 and Rrs1 are essential proteins for ribosome biogenesis. These proteins form a complex (the Rpf2-subcomplex) with 5S rRNA and two ribosomal proteins (L5 and L11). This complex is recruited to the ribosome precursor (the 90S pre-ribosome). This recruitment is necessary for the maturation of 25S rRNA. Genetic depletion of Rpf2 and Rrs1 results in accumulation of the 25S rRNA precursor. In this study, Rpf2 and Rrs1 from Aspergillus nidulans were co-overexpressed in Escherichia coli, purified and crystallized. Subsequent analysis revealed that these crystals contained the central core region of the complex consisting of both N-terminal domains. X-ray diffraction data were collected to 2.35Å resolution. Preliminary analysis revealed that the crystals belonged to space group P212121, with unit-cell parameters a = 54.1, b = 123.3, c = 133.8Å. There are two complexes in the asymmetric unit. Structure determination using selenomethionine-labelled protein is in progress.

DOI： 10.1107/S2053230X14024182

Scopus

Crystallization and preliminary X-ray crystallographic analysis of ribosome assembly factors: the Rpf2-Rrs1 complex 査読

Nozomi Asano, Akiyoshi Nakamura, Keisuke Komoda, Koji Kato, Isao Tanaka, Min Yao

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 70 ( Pt 12 ) 1649 - 1652 2014年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

Rpf2 and Rrs1 are essential proteins for ribosome biogenesis. These proteins form a complex (the Rpf2-subcomplex) with 5S rRNA and two ribosomal proteins (L5 and L11). This complex is recruited to the ribosome precursor (the 90S pre-ribosome). This recruitment is necessary for the maturation of 25S rRNA. Genetic depletion of Rpf2 and Rrs1 results in accumulation of the 25S rRNA precursor. In this study, Rpf2 and Rrs1 from Aspergillus nidulans were co-overexpressed in Escherichia coli, purified and crystallized. Subsequent analysis revealed that these crystals contained the central core region of the complex consisting of both N-terminal domains. X-ray diffraction data were collected to 2.35 angstrom resolution. Preliminary analysis revealed that the crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 54.1, b = 123.3, c = 133.8 angstrom. There are two complexes in the asymmetric unit. Structure determination using selenomethionine-labelled protein is in progress.

DOI： 10.1107/S2053230X14024182

Determination of damage-free crystal structure of an X-ray-sensitive protein using an XFEL 査読

Kunio Hirata, Kyoko Shinzawa-Itoh, Naomine Yano, Shuhei Takemura, Koji Kato, Miki Hatanaka, Kazumasa Muramoto, Takako Kawahara, Tomitake Tsukihara, Eiki Yamashita, Kensuke Tono, Go Ueno, Takaaki Hikima, Hironori Murakami, Yuichi Inubushi, Makina Yabashi, Tetsuya Ishikawa, Masaki Yamamoto, Takashi Ogura, Hiroshi Sugimoto, Jian-Ren Shen, Shinya Yoshikawa, Hideo Ago

NATURE METHODS 11 ( 7 ) 734 - U174 2014年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

We report a method of femtosecond crystallography for solving radiation damage-free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-angstrom radiation damage-free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.

DOI： 10.1038/NMETH.2962

Effective Pumping Proton Collection Facilitated by a Copper Site (CUB) of Bovine Heart Cytochrome c Oxidase, Revealed by a Newly Developed Time-resolved Infrared System 査読

Minoru Kubo, Satoru Nakashima, Satoru Yamaguchi, Takashi Ogura, Masao Mochizuki, Jiyoung Kang, Masaru Tateno, Kyoko Shinzawa-Itoh, Koji Kato, Shinya Yoshikawa

JOURNAL OF BIOLOGICAL CHEMISTRY 288 ( 42 ) 30259 - 30269 2013年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Background: Cytochrome c oxidase reduces O-2 coupled with proton pumping. Results: A newly developed time-resolved infrared system reveals transient conformational changes in the proton-pumping pathway upon CO binding to Cu-B in the O-2 reduction site. Conclusion: Cu-B promotes proton collection and effective blockage of back-leak of pumping protons. Significance: These critical findings in bioenergetics stimulate the new infrared approach for mechanistic investigation of any other protein function.
X-ray structural and mutational analyses have shown that bovine heart cytochrome c oxidase (CcO) pumps protons electrostatically through a hydrogen bond network using net positive charges created upon oxidation of a heme iron (located near the hydrogen bond network) for O-2 reduction. Pumping protons are transferred by mobile water molecules from the negative side of the mitochondrial inner membrane through a water channel into the hydrogen bond network. For blockage of spontaneous proton back-leak, the water channel is closed upon O-2 binding to the second heme (heme a(3)) after complete collection of the pumping protons in the hydrogen bond network. For elucidation of the structural bases for the mechanism of the proton collection and timely closure of the water channel, conformational dynamics after photolysis of CO (an O-2 analog)-bound CcO was examined using a newly developed time-resolved infrared system feasible for accurate detection of a single C=O stretch band of -helices of CcO in H2O medium. The present results indicate that migration of CO from heme a(3) to Cu-B in the O-2 reduction site induces an intermediate state in which a bulge conformation at Ser-382 in a transmembrane helix is eliminated to open the water channel. The structural changes suggest that, using a conformational relay system, including Cu-B, O-2, heme a(3), and two helix turns extending to Ser-382, Cu-B induces the conformational changes of the water channel that stimulate the proton collection, and senses complete proton loading into the hydrogen bond network to trigger the timely channel closure by O-2 transfer from Cu-B to heme a(3).

DOI： 10.1074/jbc.M113.473983