Faculty Profiles - KATOU Kouji

Papers - KATOU Kouji

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The C-terminal helix of ribosomal P stalk recognizes a hydrophobic groove of elongation factor 2 in a novel fashion. Reviewed

Tanzawa T, Kato K, Girodat D, Ose T, Kumakura Y, Wieden HJ, Uchiumi T, Tanaka I, Yao M

Nucleic acids research 46 ( 6 ) 3232 - 3244 2018.4

Biochemical and structural characterization of Marinomonas mediterranea D-mannose isomerase Marme_2490 phylogenetically distant from known enzymes Reviewed

Wataru Saburi, Nongluck Jaito, Koji Kato, Yuka Tanaka, Min Yao, Haruhide Mori

Biochimie 144 63 - 73 2018.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier B.V.

D-Mannose isomerase (MI) reversibly isomerizes D-mannose to D-fructose, and is attractive for producing D-mannose from inexpensive D-fructose. It belongs to the N-acylglucosamine 2-epimerase (AGE) superfamily along with AGE, cellobiose 2-epimerase (CE), and aldose-ketose isomerase (AKI). In this study, Marinomonas mediterranea Marme_2490, showing low sequence identity with any known enzymes, was found to isomerize D-mannose as its primary substrate. Marme_2490 also isomerized D-lyxose and 4-OH D-mannose derivatives (D-talose and 4-O-monosaccharyl-D-mannose). Its activity for D-lyxose is known in other D-mannose isomerizing enzymes, such as MI and AKI, but we identified, for the first time, its activity for 4-OH D-mannose derivatives. Marme_2490 did not isomerize D-glucose, as known MIs do not, while AKI isomerizes both D-mannose and D-glucose. Thus, Marme_2490 was concluded to be an MI. The initial and equilibrium reaction products were analyzed by NMR to illuminate mechanistic information regarding the Marme_2490 reaction. The analysis of the initial reaction product revealed that β-D-mannose was formed. In the analysis of the equilibrated reaction products in D2O, signals of 2-H of D-mannose and 1-H of D-fructose were clearly detected. This indicates that these protons are not substituted with deuterium from D2O and Marme_2490 catalyzes the intramolecular proton transfer between 1-C and 2-C. The crystal structure of Marme_2490 in a ligand-free form was determined and found that Marme_2490 is formed by an (α/α)6-barrel, which is commonly observed in AGE superfamily enzymes. Despite diverse reaction specificities, the orientations of residues involved in catalysis and substrate binding by Marme_2490 were similar to those in both AKI (Salmonella enterica AKI) and epimerase (Rhodothermus marinus CE). The Marme_2490 structure suggested that the α7→α8 and α11→α12 loops of the catalytic domain participated in the formation of an open substrate-binding site to provide sufficient space to bind 4-OH D-mannose derivatives.

DOI： 10.1016/j.biochi.2017.10.016

Structural basis for tRNA-dependent cysteine biosynthesis Reviewed

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 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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 beta-L-arabinopyranosidase/alpha-galactosidase from Bacteroides thetaiotaomicron Reviewed

Asako Kikuchi, Masayuki Okuyama, Koji Kato, Shohei Osaki, Min Ma, Yuya Kumagai, Kana Matsunaga, Patcharapa Kiahan, Takayoshi Tagami, Min Yao, Atsuo Kimura

BIOCHIMIE 142 41 - 50 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, alpha-glucoside hydrolase and agalactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 a-glucoside hydrolase and alpha-galactosidase, were characterized in this study. BT3664 was identified to be an a-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both beta-L-arabinopyranoside and alpha-D-galactopyranoside, and thus we designate BT3661 as a beta-L-arabinopyranosidase/alpha-D-galactosidase, Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 agalactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears G1u361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The k(cat)/K-m value for the hydrolysis of p-nitrophenyl a-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the kcat/Km value for hydrolysis of p-nitrophenyl beta-D-arabinopyranoside. (C) 2017 Published by Elsevier B.V.

DOI： 10.1016/j.biochi.2017.08.003

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

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

SCIENTIFIC REPORTS 7 ( 1 ) 13018 2017.10

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 2016.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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

SCIENCE ADVANCES 2 ( 3 ) e1501397 2016.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 2016.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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 angstrom Resolution Reviewed

Zuoqi Gai, Asuka Matsuno, Koji Kato, Sanae Kato, Md Rafiqul Islam Khan, Takeshi Shimizu, Takeya Yoshioka, Yuki Kato, Hideki Kishimura, Gaku Kanno, Yoshikatsu Miyabe, Tohru Terada, Yoshikazu Tanaka, Min Yao

STRUCTURE 23 ( 12 ) 2204 - 2212 2015.12

Language：English Publishing type：Research paper (scientific journal) Publisher：CELL PRESS

Molluscan hemocyanin, a copper-containing oxygen transporter, is one of the largest known proteins. Although molluscan hemocyanins are currently applied as immunotherapeutic agents, their precise structure has not been determined because of their enormous size. Here, we have determined the first X-ray crystal structure of intact molluscan hemocyanin. The structure unveiled the architecture of the 3.8-MDa supermolecule composed of homologous functional units (FUs), wherein the dimers of FUs hierarchically associated to form the entire cylindrical decamer. Most of the specific inter-FU interactions were localized at narrow regions in the FU dimers, suggesting that rigid FU dimers formed by specific interactions assemble with flexibility. Furthermore, the roles of carbohydrates in assembly and allosteric effect, and conserved sulfur-containing residues in copper incorporation, were revealed. The precise structural information obtained in this study will accelerate our understanding of the molecular basis of hemocyanin and its future applications.

DOI： 10.1016/j.str.2015.09.008

Structural basis for pore-forming mechanism of staphylococcal alpha-hemolysin Reviewed

Takaki Sugawara, Daichi Yamashita, Koji Kato, Zhao Peng, Junki Ueda, Jun Kaneko, Yoshiyuki Kamio, Yoshikazu Tanaka, Min Yao

TOXICON 108 226 - 231 2015.12

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Staphylococcal alpha-hemolysin (alpha-HL) is a beta-barrel pore-forming toxin (PFT) expressed by Staphylococcus aureus. alpha-HL is secreted as a water-soluble monomeric protein, which binds to target membranes and forms membrane-inserted heptameric pores. To explore the pore-forming mechanism of alpha-HL in detail, we determined the crystal structure of the alpha-HL monomer and prepore using H35A mutant and W179A/R200A mutant, respectively. Although the overall structure of the monomer was similar to that of other staphylococcal PFTs, a marked difference was observed in the N-terminal amino latch, which bent toward the prestem. Moreover, the prestem was fastened by the cap domain with a key hydrogen bond between Asp45 and Tyr118. Prepore structure showed that the transmembrane region is roughly formed with flexibility, although the upper half of the beta-barrel is formed appropriately. Structure comparison among monomer, prepore and pore revealed a series of motions, in which the N-terminal amino latch released upon oligomerization destroys its own key hydrogen bond between Asp45 Tyr118. This action initiated the protrusion of the prestem. Y118F mutant and the N-terminal truncated mutant markedly decreased in the hemolytic activity, indicating the importance of the key hydrogen bond and the N-terminal amino latch on the pore formation. Based on these observations, we proposed a dynamic molecular mechanism of pore formation for alpha-HL. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.toxicon.2015.09.033

Structural analysis of the alpha-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism Reviewed

Xing Shen, Wataru Saburi, Zuoqi Gai, Koji Kato, Teruyo Ojima-Kato, Jian Yu, Keisuke Komoda, Yusuke Kido, Hirokazu Matsui, Haruhide Mori, Min Yao

ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 71 ( Pt 6 ) 1382 - 1391 2015.6

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

alpha-Glucosidases, which catalyze the hydrolysis of the alpha-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of alpha-glucosides. Halomonas sp. H11 alpha-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the alpha-(1 -> 4)-linked disaccharide maltose among naturally occurring substrates. Although several three- dimensional structures of GH13 members have been solved, the disaccharide specificity and alpha-(1 -> 4) recognition mechanism of alpha-glucosidase are unclear owing to a lack of corresponding substrate- bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl- enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with d- glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long beta ->alpha loop 4 which exists in alpha-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite + 1. Furthermore, an explanation of the alpha-glucosidase reaction mechanism is proposed based on the glucosyl- enzyme intermediate structure.

DOI： 10.1107/S139900471500721X

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

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 2015.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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

NUCLEIC ACIDS RESEARCH 43 ( 9 ) 4746 - 4757 2015.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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

FEBS LETTERS 589 ( 4 ) 484 - 489 2015.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 2015.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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

Acta Crystallographica Section F:Structural Biology Communications 70 1649 - 1652 2014.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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

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

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 70 ( Pt 12 ) 1649 - 1652 2014.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 2014.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 2013.10

Language：English Publishing type：Research paper (scientific journal) Publisher：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

精密結晶構造解析によるチトクロム酸化酵素の反応機構の研究

矢野直峰, 新澤-伊藤恭子, 畑中美紀, 武村修平, 藤澤秀徳, 村本和優, 加藤公児, 山下栄樹, 吉川信也, 月原冨武

日本結晶学会誌 54 ( 0 ) s19 - s19 2012

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