Faculty Profiles - KATOU Kouji

Papers - KATOU Kouji

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Biochemical and structural analysis of the mechanism for the catalysis and specificity of cellobiose 2-epimerase from Rhodothermus marinus. Reviewed International journal

Wataru Saburi, Hirohiko Muto-Fukiya, Nongluck Jaito, Koji Kato, Jian Yu, Min Yao, Haruhide Mori

Bioscience, biotechnology, and biochemistry 2025.3

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

Cellobiose 2-epimerase (CE) catalyzes C-2 epimerization of reducing end d-glucose/d-mannose residue of β-(1→4)-disaccharides, and also slightly catalyzes aldose-ketose conversion. In this study, we investigated the structure-function relationship of Rhodothermus marinus CE (RmCEs). In 2H2O, 2H replaced the 2-H of the reducing end sugar residue, suggesting a proton abstraction-addition mechanism via the cis-enediolate intermediate. The structure of the RmCE-mannobiitol complex showed that His259 was suitable for abstracting 2-H from d-mannose residue, whereas His390 was suitable for the d-glucose residue. H259A and H390A mutations abolished activity for Galβ1-4Man and Galβ1-4Glc formation from Galβ1-4Fru, respectively, and these mutants catalyzed both epimerization and isomerization to Galβ1-4Glc and Galβ1-4Man, respectively. Ala substitution of the residues interacting with the 2-O of the reducing end sugar residue significantly reduced the velocity for epimerization, but not for isomerization. Trp385, stacked onto the non-reducing-end sugar residues of disaccharides, was shown to be important for disaccharide specificity.

DOI： 10.1093/bbb/zbaf042

Structural study of the chlorophyll between Lhca8 and PsaJ in an Antarctica green algal photosystem I-LHCI supercomplex revealed by its atomic structure Reviewed

Pi-Cheng Tsai, Koji Kato, Jian-Ren Shen, Fusamichi Akita

Biochimica et Biophysica Acta (BBA) - Bioenergetics 149543 - 149543 2025.2

Biochemical evidence for the diversity of LHCI proteins in PSI-LHCI from the red alga Galdieria sulphuraria NIES-3638. Reviewed International journal

Ryo Nagao, Haruya Ogawa, Takehiro Suzuki, Naoshi Dohmae, Koji Kato, Yoshiki Nakajima, Jian-Ren Shen

Photosynthesis research 163 ( 1 ) 14 - 14 2025.1

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

Red algae are photosynthetic eukaryotes whose light-harvesting complexes (LHCs) associate with photosystem I (PSI). In this study, we examined characteristics of PSI-LHCI, PSI, and LHCI isolated from the red alga Galdieria sulphuraria NIES-3638. The PSI-LHCI supercomplexes were purified using anion-exchange chromatography followed by hydrophobic-interaction chromatography, and finally by trehalose density gradient centrifugation. PSI and LHCI were similarly prepared following the dissociation of PSI-LHCI with Anzergent 3-16. Polypeptide analysis of PSI-LHCI revealed the presence of PSI and LHC proteins, along with red-lineage chlorophyll a/b-binding-like protein (RedCAP), which is distinct from LHC proteins within the LHC protein superfamily. RedCAP, rather than LHC proteins, exhibited tight binding to PSI. Carotenoid analysis of LHCI identified zeaxanthin, β-cryptoxanthin, and β-carotene, with zeaxanthin particularly enriched, which is consistent with other red algal LHCIs. A Qy peak of chlorophyll a in the LHCI absorption spectrum was blue-shifted compared with those of PSI-LHCI and PSI, and a fluorescence emission peak was similarly shifted to shorter wavelengths. Based on these results, we discuss the diversity of LHC proteins and RedCAP in red algal PSI-LHCI supercomplexes.

DOI： 10.1007/s11120-024-01134-1

Crystallographic analysis of the Escherichia coli tRNA seleno-modification enzyme in complex with tRNA Reviewed

Takuya Usui, Sayaka Ono, Akiyoshi Nakamura, Koji Kato, Toyoyuki Ose, Min Yao

Acta Crystallographica Section F Structural Biology Communications 81 ( 2 ) 2025.1

Publishing type：Research paper (scientific journal) Publisher：International Union of Crystallography (IUCr)

The bacterial enzyme tRNA 2-selenouridine synthase (SelU) catalyzes the conversion of 5-substituted 2-thiouridine (R5S2U) to 5-substituted 2-selenouridine (R5Se2U) at the wobble positions of several tRNAs. Seleno-modification potentially regulates translation efficiency in response to selenium availability. Notably, SelU uses the 2-geranylthiouridine (R5geS2U) intermediate for sulfur removal, and this geranylthiol (geS) is a unique leaving group among tRNA-maturation enzymes. However, the underlying sequence of the SelU reaction remains unclear. Here, a crystallographic study of the Escherichia coli SelU–tRNA complex is reported. Robust and well formed SelU–tRNA crystals were obtained after several optimizations, including co-expression with tRNA and additive screening. Diffraction data were collected at a resolution of 3.10 Å using a wavelength of 1.0000 Å. The crystals belonged to space group C2, and the phase was determined by molecular replacement using the AlphaFold2-predicted SelU structure as a search model. Electron-density mapping revealed the presence of two SelU–tRNA complexes in the asymmetric unit.

DOI： 10.1107/s2053230x25000044

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex Reviewed

Koji Kato, Yoshiki Nakajima, Jian Xing, Minoru Kumazawa, Haruya Ogawa, Jian-Ren Shen, Kentaro Ifuku, Ryo Nagao

eLife 13 2024.10

Authorship：Lead author Publishing type：Research paper (scientific journal) Publisher：eLife Sciences Publications, Ltd

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein–protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

DOI： 10.7554/elife.99858.3

Other Link： https://cdn.elifesciences.org/articles/99858/elife-99858-v1.xml

Presence of low-energy chlorophylls d in photosystem I trimer and monomer cores isolated from Acaryochloris sp. NBRC 102871. Reviewed International journal

Ryo Nagao, Haruki Yamamoto, Haruya Ogawa, Hibiki Ito, Yuma Yamamoto, Takehiro Suzuki, Koji Kato, Yoshiki Nakajima, Naoshi Dohmae, Jian-Ren Shen

Photosynthesis research 161 ( 3 ) 203 - 212 2024.9

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

Acaryochloris species belong to a special category of cyanobacteria possessing chlorophyll (Chl) d. One of the photosynthetic characteristics of Acaryochloris marina MBIC11017 is that the absorption spectra of photosystem I (PSI) showed almost no bands and shoulders of low-energy Chls d over 740 nm. In contrast, the absorption spectra of other Acaryochloris species showed a shoulder around 740 nm, suggesting that low-energy Chls d within PSI are diversified among Acaryochloris species. In this study, we purified PSI trimer and monomer cores from Acaryochloris sp. NBRC 102871 and examined their protein and pigment compositions and spectral properties. The protein bands and pigment compositions of the PSI trimer and monomer of NBRC102871 were virtually identical to those of MBIC11017. The absorption spectra of the NBRC102871 PSIs exhibited a shoulder around 740 nm, whereas the fluorescence spectra of PSI trimer and monomer displayed maximum peaks at 754 and 767 nm, respectively. These spectral properties were different from those of MBIC11017, indicating the presence of low-energy Chls d within the NBRC102871 PSIs. Moreover, we analyzed the NBRC102871 genome to identify amino acid sequences of PSI proteins and compared them with those of the A. marina MBIC11017 and MBIC10699 strains whose genomes are available. The results showed that some of the sequences in NBRC102871 were distinct from those in MBIC11017 and MBIC10699. These findings provide insights into the variety of low-energy Chls d with respect to the protein environments of PSI cores among the three Acaryochloris strains.

DOI： 10.1007/s11120-024-01108-3

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs. Reviewed International journal

Koji Kato, Tasuku Hamaguchi, Minoru Kumazawa, Yoshiki Nakajima, Kentaro Ifuku, Shunsuke Hirooka, Yuu Hirose, Shin-Ya Miyagishima, Takehiro Suzuki, Keisuke Kawakami, Naoshi Dohmae, Koji Yonekura, Jian-Ren Shen, Ryo Nagao

Proceedings of the National Academy of Sciences of the United States of America 121 ( 11 ) e2319658121 2024.3

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

DOI： 10.1073/pnas.2319658121

Oxygen-evolving photosystem II structures during S1–S2–S3 transitions Reviewed

Hongjie Li, Yoshiki Nakajima, Eriko Nango, Shigeki Owada, Daichi Yamada, Kana Hashimoto, Fangjia Luo, Rie Tanaka, Fusamichi Akita, Koji Kato, Jungmin Kang, Yasunori Saitoh, Shunpei Kishi, Huaxin Yu, Naoki Matsubara, Hajime Fujii, Michihiro Sugahara, Mamoru Suzuki, Tetsuya Masuda, Tetsunari Kimura, Tran Nguyen Thao, Shinichiro Yonekura, Long-Jiang Yu, Takehiko Tosha, Kensuke Tono, Yasumasa Joti, Takaki Hatsui, Makina Yabashi, Minoru Kubo, So Iwata, Hiroshi Isobe, Kizashi Yamaguchi, Michihiro Suga, Jian-Ren Shen

Nature 626 ( 7999 ) 670 - 677 2024.1

Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Abstract

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of S_i states (i = 0–4) at the Mn₄CaO₅ cluster^1–3, during which an extra oxygen (O6) is incorporated at the S₃ state to form a possible dioxygen^4–7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). Y_Z, a tyrosine residue that connects the reaction centre P680 and the Mn₄CaO₅ cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca²⁺ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O–O bond formation.

DOI： 10.1038/s41586-023-06987-5

Other Link： https://www.nature.com/articles/s41586-023-06987-5

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin. Reviewed International journal

Yukino Sato, Tsubasa Hashimoto, Koji Kato, Akiko Okamura, Kaito Hasegawa, Tsukasa Shinone, Yoshikazu Tanaka, Yoshiki Tanaka, Tomoya Tsukazaki, Takashi Tsukamoto, Makoto Demura, Min Yao, Takashi Kikukawa

The Journal of biological chemistry 299 ( 12 ) 105393 - 105393 2023.12

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

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

DOI： 10.1016/j.jbc.2023.105393

Structural basis of EHEP-mediated offense against phlorotannin-induced defense from brown algae to protect akuBGL activity. Reviewed International journal

Xiaomei Sun, Yuxin Ye, Naofumi Sakurai, Hang Wang, Koji Kato, Jian Yu, Keizo Yuasa, Akihiko Tsuji, Min Yao

eLife 12 2023.11

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

The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai, which uses β-glucosidase (akuBGL) to digest the laminarin in algae into glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect akuBGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of the apo and tannic acid (TNA, a phlorotannin analog) bound forms of EHEP, as well as the apo akuBGL. EHEP consisted of three peritrophin-A domains arranged in a triangular shape and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP-TNA led us to find that EHEP can be resolubilized from phlorotannin precipitation at an alkaline pH, which reflects a requirement in the digestive tract. akuBGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits akuBGL by occupying the substrate-binding pocket, and EHEP protects akuBGL against this inhibition by binding with phlorotannin to free the akuBGL pocket.

DOI： 10.7554/eLife.88939

Tight association of CpcL with photosystem I in Anabaena sp. PCC 7120 grown under iron-deficient conditions. Reviewed International journal

Shota Shimizu, Haruya Ogawa, Naoki Tsuboshita, Takehiro Suzuki, Koji Kato, Yoshiki Nakajima, Naoshi Dohmae, Jian-Ren Shen, Ryo Nagao

Biochimica et biophysica acta. Bioenergetics 148993 - 148993 2023.6

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

Phycobilisomes (PBSs), which are huge pigment-protein complexes displaying distinctive color variations, bind to photosystem cores for excitation-energy transfer. It is known that the isolation of supercomplexes consisting of PBSs and photosystem I (PSI) or PBSs and photosystem II (PSII) is challenging due to weak interactions between PBSs and the photosystem cores. In this study, we succeeded in purifying PSI-monomer-PBS and PSI-dimer-PBS supercomplexes from the cyanobacterium Anabaena sp. PCC 7120 grown under iron-deficient conditions by anion-exchange chromatography, followed by trehalose density gradient centrifugation. The absorption spectra of the two types of supercomplexes showed apparent bands originating from PBSs, and their fluorescence-emission spectra exhibited characteristic peaks of PBSs. Two-dimensional blue-native (BN)/SDS-PAGE of the two samples showed a band of CpcL, which is a linker protein of PBS, in addition to PsaA/B subunits. Since interactions of PBSs with PSI are easily dissociated during BN-PAGE using thylakoids from this cyanobacterium grown under iron-replete conditions, it is suggested that iron deficiency for Anabaena induces tight associations of CpcL with PSI, resulting in the formation of PSI-monomer-PBS and PSI-dimer-PBS supercomplexes. Based on these findings, we discuss interactions of PBSs with PSI in Anabaena in response to growth conditions.

DOI： 10.1016/j.bbabio.2023.148993

Isolation and characterization of trimeric and monomeric PSI cores from Acaryochloris marina MBIC11017 Reviewed

Ryo Nagao, Haruya Ogawa, Naoki Tsuboshita, Koji Kato, Reona Toyofuku, Tatsuya Tomo, Jian-Ren Shen

Photosynthesis Research 2023.5

Identification and Analysis of Monoclonal Antibodies with Neutralizing Activity against Diverse SARS-CoV-2 Variants Reviewed

Hanako Ishimaru, Mitsuhiro Nishimura, Lidya Handayani Tjan, Silvia Sutandhio, Maria Istiqomah Marini, Gema Barlian Effendi, Hideki Shigematsu, Koji Kato, Natsumi Hasegawa, Kaito Aoki, Yukiya Kurahashi, Koichi Furukawa, Mai Shinohara, Tomoka Nakamura, Jun Arii, Tatsuya Nagano, Sachiko Nakamura, Shigeru Sano, Sachiyo Iwata, Shinya Okamura, Yasuko Mori

Journal of Virology 2023.5

Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Omicron variants of SARS-CoV-2 acquired escape ability from host immunity and authorized antibody therapeutics and thereby have been spreading worldwide. We reported that patients infected with an early SARS-CoV-2 variant, D614G, and who received subsequent two-dose mRNA vaccination have high neutralizing antibody titer against Omicron lineages.

DOI： 10.1128/jvi.00286-23

Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on β→α Loop 5 Reviewed

Waraporn Auiewiriyanukul, Wataru Saburi, Tomoya Ota, Jian Yu, Koji Kato, Min Yao, Haruhide Mori

Molecules 28 ( 7 ) 3109 - 3109 2023.3

Publishing type：Research paper (scientific journal) Publisher：MDPI AG

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on β→α loop 5 of the catalytic (β/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.

DOI： 10.3390/molecules28073109

Function and structure of <i>Lacticaseibacillus casei</i> GH35 β-galactosidase LBCZ_0230 with high hydrolytic activity to lacto-<i>N</i>-biose I and galacto-<i>N</i>-biose Reviewed

Saburi Wataru, Tomoya Ota, Koji Kato, Takayoshi Tagami, Keitaro Yamashita, Min Yao, Haruhide Mori

Journal of Applied Glycoscience 2023.3

Structure of a monomeric photosystem I core associated with iron-stress-induced-A proteins from Anabaena sp. PCC 7120. Reviewed International journal

Ryo Nagao, Koji Kato, Tasuku Hamaguchi, Yoshifumi Ueno, Naoki Tsuboshita, Shota Shimizu, Miyu Furutani, Shigeki Ehira, Yoshiki Nakajima, Keisuke Kawakami, Takehiro Suzuki, Naoshi Dohmae, Seiji Akimoto, Koji Yonekura, Jian-Ren Shen

Nature communications 14 ( 1 ) 920 - 920 2023.2

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions. The cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, their binding property and functional roles in PSI are still missing. We analyzed a cryo-electron microscopy structure of a PSI-IsiA supercomplex isolated from Anabaena grown under an iron-deficient condition. The PSI-IsiA structure contains six IsiA subunits associated with the PsaA side of a PSI core monomer. Three of the six IsiA subunits were identified as IsiA1 and IsiA2. The PSI-IsiA structure lacks a PsaL subunit; instead, a C-terminal domain of IsiA2 occupies the position of PsaL, which inhibits the oligomerization of PSI, leading to the formation of a PSI monomer. Furthermore, excitation-energy transfer from IsiAs to PSI appeared with a time constant of 55 ps. These findings provide insights into both the molecular assembly of the Anabaena IsiA family and the functional roles of IsiAs.

DOI： 10.1038/s41467-023-36504-1

Biochemical and spectroscopic characterization of PSI-LHCI from the red alga Cyanidium caldarium. Reviewed International journal

Ryo Nagao, Yoshifumi Ueno, Miyu Furutani, Koji Kato, Jian-Ren Shen, Seiji Akimoto

Photosynthesis research 2023.2

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

Light-harvesting complexes (LHCs) have been diversified in oxygenic photosynthetic organisms, and play an essential role in capturing light energy which is transferred to two types of photosystem cores to promote charge-separation reactions. Red algae are one of the groups of photosynthetic eukaryotes, and their chlorophyll (Chl) a-binding LHCs are specifically associated with photosystem I (PSI). In this study, we purified three types of preparations, PSI-LHCI supercomplexes, PSI cores, and isolated LHCIs, from the red alga Cyanidium caldarium, and examined their properties. The polypeptide bands of PSI-LHCI showed characteristic PSI and LHCI components without contamination by other proteins. The carotenoid composition of LHCI displayed zeaxanthins, β-cryptoxanthins, and β-carotenes. Among the carotenoids, zeaxanthins were enriched in LHCI. On the contrary, both zeaxanthins and β-cryptoxanthins could not be detected from PSI, suggesting that zeaxanthins and β-cryptoxanthins are bound to LHCI but not PSI. A Qy peak of Chl a in the absorption spectrum of LHCI was shifted to a shorter wavelength than those in PSI and PSI-LHCI. This tendency is in line with the result of fluorescence-emission spectra, in which the emission maxima of PSI-LHCI, PSI, and LHCI appeared at 727, 719, and 677 nm, respectively. Time-resolved fluorescence spectra of LHCI represented no 719 and 727-nm fluorescence bands from picoseconds to nanoseconds. These results indicate that energy levels of Chls around/within LHCIs and within PSI are changed by binding LHCIs to PSI. Based on these findings, we discuss the expression, function, and structure of red algal PSI-LHCI supercomplexes.

DOI： 10.1007/s11120-023-00999-y

Novel monoclonal antibodies showing broad neutralizing activity for SARS-CoV-2 variants including Omicrons BA.5 and BA.2.75

Hanako Ishimaru, Mitsuhiro Nishimura, Lidya Handayani Tjan, Silvia Sutandhio, Maria Istiqomah Marini, Gema Barlian Effendi, Hideki Shigematsu, Koji Kato, Natsumi Hasegawa, Kaito Aoki, Yukiya Kurahashi, Koichi Furukawa, Mai Shinohara, Tomoka Nakamura, Jun Arii, Tatsuya Nagano, Sachiko Nakamura, Shigeru Sano, Sachiyo Iwata, Yasuko Mori

2022.9

Publisher：Cold Spring Harbor Laboratory

Summary

We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and BA.5. Our findings confirm that the wildtype-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants (including BA.5 and BA.2.75). The monoclonal antibodies obtained herein could serve as novel prophylaxis and therapeutics against not only current SARS-CoV-2 viruses but also future variants that may arise.

DOI： 10.1101/2022.09.02.506305

Excited-state intermediates in a designer protein encoding a phototrigger caught by an X-ray free-electron laser. Reviewed International journal

Xiaohong Liu, Pengcheng Liu, Hongjie Li, Zhen Xu, Lu Jia, Yan Xia, Minling Yu, Wenqin Tang, Xiaolei Zhu, Chao Chen, Yuanlin Zhang, Eriko Nango, Rie Tanaka, Fangjia Luo, Koji Kato, Yoshiki Nakajima, Shunpei Kishi, Huaxin Yu, Naoki Matsubara, Shigeki Owada, Kensuke Tono, So Iwata, Long-Jiang Yu, Jian-Ren Shen, Jiangyun Wang

Nature chemistry 14 ( 9 ) 1054 - 1060 2022.9

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

One of the primary objectives in chemistry research is to observe atomic motions during reactions in real time. Although X-ray free-electron lasers (XFELs) have facilitated the capture of reaction intermediates using time-resolved serial femtosecond crystallography (TR-SFX), only a few natural photoactive proteins have been investigated using this method, mostly due to the lack of suitable phototriggers. Here we report the genetic encoding of a xanthone amino acid (FXO), as an efficient phototrigger, into a rationally designed human liver fatty-acid binding protein mutant (termed XOM), which undergoes photo-induced C-H bond transformation with high selectivity and quantum efficiency. We solved the structures of XOM before and 10-300 ns after flash illumination, at 1.55-1.70 Å resolutions, and captured the elusive excited-state intermediates responsible for precise C-H bond activation. We expect that most redox enzymes can now be investigated by TR-SFX, using our method, to reveal reaction intermediates key for their efficiency and selectivity.

DOI： 10.1038/s41557-022-00992-3

Structural basis for the absence of low-energy chlorophylls in a photosystem I trimer from Gloeobacter violaceus. Reviewed International journal

Koji Kato, Tasuku Hamaguchi, Ryo Nagao, Keisuke Kawakami, Yoshifumi Ueno, Takehiro Suzuki, Hiroko Uchida, Akio Murakami, Yoshiki Nakajima, Makio Yokono, Seiji Akimoto, Naoshi Dohmae, Koji Yonekura, Jian-Ren Shen

eLife 11 2022.4

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Photosystem I (PSI) is a multi-subunit pigment-protein complex that functions in light-harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation in photosynthetic organisms. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the sites of low-energy Chls are still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primordial cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure shows the absence of some subunits commonly found in other cyanobacteria, confirming the primordial nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 is missing in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. These findings provide insights into not only the identity of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls in oxyphototrophs.

DOI： 10.7554/eLife.73990

Structural basis for different types of hetero-tetrameric light-harvesting complexes in a diatom PSII-FCPII supercomplex. Reviewed International journal

Ryo Nagao, Koji Kato, Minoru Kumazawa, Kentaro Ifuku, Makio Yokono, Takehiro Suzuki, Naoshi Dohmae, Fusamichi Akita, Seiji Akimoto, Naoyuki Miyazaki, Jian-Ren Shen

Nature communications 13 ( 1 ) 1764 - 1764 2022.4

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Fucoxanthin chlorophyll (Chl) a/c-binding proteins (FCPs) function as light harvesters in diatoms. The structure of a diatom photosystem II-FCPII (PSII-FCPII) supercomplex have been solved by cryo-electron microscopy (cryo-EM) previously; however, the FCPII subunits that constitute the FCPII tetramers and monomers are not identified individually due to their low resolutions. Here, we report a 2.5 Å resolution structure of the PSII-FCPII supercomplex using cryo-EM. Two types of tetrameric FCPs, S-tetramer, and M-tetramer, are identified as different types of hetero-tetrameric complexes. In addition, three FCP monomers, m1, m2, and m3, are assigned to different gene products of FCP. The present structure also identifies the positions of most Chls c and diadinoxanthins, which form a complicated pigment network. Excitation-energy transfer from FCPII to PSII is revealed by time-resolved fluorescence spectroscopy. These structural and spectroscopic findings provide insights into an assembly model of FCPII and its excitation-energy transfer and quenching processes.

DOI： 10.1038/s41467-022-29294-5

Structure of a tetrameric photosystem I from a glaucophyte alga Cyanophora paradoxa. Reviewed International journal

Koji Kato, Ryo Nagao, Yoshifumi Ueno, Makio Yokono, Takehiro Suzuki, Tian-Yi Jiang, Naoshi Dohmae, Fusamichi Akita, Seiji Akimoto, Naoyuki Miyazaki, Jian-Ren Shen

Nature communications 13 ( 1 ) 1679 - 1679 2022.3

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Photosystem I (PSI) is one of the two photosystems functioning in light-energy harvesting, transfer, and electron transfer in photosynthesis. However, the oligomerization state of PSI is variable among photosynthetic organisms. We present a 3.8-Å resolution cryo-electron microscopic structure of tetrameric PSI isolated from the glaucophyte alga Cyanophora paradoxa, which reveals differences with PSI from other organisms in subunit composition and organization. The PSI tetramer is organized in a dimer of dimers with a C2 symmetry. Unlike cyanobacterial PSI tetramers, two of the four monomers are rotated around 90°, resulting in a completely different pattern of monomer-monomer interactions. Excitation-energy transfer among chlorophylls differs significantly between Cyanophora and cyanobacterial PSI tetramers. These structural and spectroscopic features reveal characteristic interactions and excitation-energy transfer in the Cyanophora PSI tetramer, suggesting that the Cyanophora PSI could represent a turning point in the evolution of PSI from prokaryotes to eukaryotes.

DOI： 10.1038/s41467-022-29303-7

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer. Reviewed International journal

Huaxin Yu, Tasuku Hamaguchi, Yoshiki Nakajima, Koji Kato, Keisuke Kawakami, Fusamichi Akita, Koji Yonekura, Jian-Ren Shen

Biochimica et biophysica acta. Bioenergetics 1862 ( 10 ) 148471 - 148471 2021.10

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

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.

DOI： 10.1016/j.bbabio.2021.148471

Structural basis for the absence of low-energy chlorophylls responsible for photoprotection from a primitive cyanobacterial PSI

Koji Kato, Tasuku Hamaguchi, Ryo Nagao, Keisuke Kawakami, Yoshifumi Ueno, Takehiro Suzuki, Hiroko Uchida, Akio Murakami, Yoshiki Nakajima, Makio Yokono, Seiji Akimoto, Naoshi Dohmae, Koji Yonekura, Jian-Ren Shen

2021.10

Publisher：Cold Spring Harbor Laboratory

Abstract

Photosystem I (PSI) of photosynthetic organisms is a multi-subunit pigment-protein complex and functions in light harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO₂ fixation. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the site of low-energy Chls is still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primitive cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure showed absence of some subunits commonly found in other cyanobacteria, confirming the primitive nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 does not exist in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. Since Gloeobacter is susceptible to light, this indicates that Low1 serves as a main photoprotection site in most oxyphototrophs, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs. Thus, these findings provide insights into not only the functional significance of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls responsible for the photoprotection machinery from photosynthetic prokaryotes to eukaryotes.

DOI： 10.1101/2021.09.29.462462

High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams. Reviewed International journal

Koji Kato, Naoyuki Miyazaki, Tasuku Hamaguchi, Yoshiki Nakajima, Fusamichi Akita, Koji Yonekura, Jian-Ren Shen

Communications biology 4 ( 1 ) 382 - 382 2021.3

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

Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state and its final state structure has not been solved. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, and reducing the beam dosage by reducing frames from 50 to 2 yielded a similar resolution but reduced the damage remarkably. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

DOI： 10.1038/s42003-021-01919-3

Molecular organizations and function of iron-stress-induced-A protein family in Anabaena sp. PCC 7120. Reviewed International journal

Ryo Nagao, Makio Yokono, Yoshifumi Ueno, Takehiro Suzuki, Koji Kato, Ka-Ho Kato, Naoki Tsuboshita, Tian-Yi Jiang, Naoshi Dohmae, Jian-Ren Shen, Shigeki Ehira, Seiji Akimoto

Biochimica et biophysica acta. Bioenergetics 1862 ( 1 ) 148327 - 148327 2021.1

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

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions, and surround photosystem I (PSI) trimer with a ring formation. A cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, it is unknown how the IsiAs are associated with PSI. Here we report on molecular organizations and function of the IsiAs in this cyanobacterium. A deletion mutant of the isiA1 gene was constructed, and the four types of thylakoids were prepared from the wild-type (WT) and ΔisiA1 cells under iron-replete (+Fe) and iron-deficient (-Fe) conditions. Immunoblotting analysis exhibits a clear expression of the IsiA1 in the WT-Fe. The PSI-IsiA1 supercomplex is found in the WT-Fe, and excitation-energy transfer from IsiA1 to PSI is verified by time-resolved fluorescence analyses. Instead of the IsiA1, both IsiA2 and IsiA3 are bound to PSI monomer in the ΔisiA1-Fe. These findings provide insights into multiple-expression system of the IsiA family in this cyanobacterium.

DOI： 10.1016/j.bbabio.2020.148327

High-resolution cryo-EM structure of photosystem II: Effects of electron beam damage

Koji Kato, Naoyuki Miyazaki, Tasuku Hamaguchi, Yoshiki Nakajima, Fusamichi Akita, Koji Yonekura, Jian-Ren Shen

2020.10

Publisher：Cold Spring Harbor Laboratory

Abstract

Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state, and its final state structure has not been solved because of the low efficiencies of the S-state transitions in the crystals. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, which was reduced by reducing the electron dose. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

DOI： 10.1101/2020.10.18.344648

A solution-free crystal-mounting platform for native SAD. Reviewed International journal

Jian Yu, Akira Shinoda, Koji Kato, Isao Tanaka, Min Yao

Acta crystallographica. Section D, Structural biology 76 ( Pt 10 ) 938 - 945 2020.10

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

The native SAD phasing method uses the anomalous scattering signals from the S atoms contained in most proteins, the P atoms in nucleic acids or other light atoms derived from the solution used for crystallization. These signals are very weak and careful data collection is required, which makes this method very difficult. One way to enhance the anomalous signal is to use long-wavelength X-rays; however, these wavelengths are more strongly absorbed by the materials in the pathway. Therefore, a crystal-mounting platform for native SAD data collection that removes solution around the crystals has been developed. This platform includes a novel solution-free mounting tool and an automatic robot, which extracts the surrounding solution, flash-cools the crystal and inserts the loop into a UniPuck cassette for use in the synchrotron. Eight protein structures (including two new structures) have been successfully solved by the native SAD method from crystals prepared using this platform.

DOI： 10.1107/S2059798320011584

A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes Reviewed

Hajime Wakui, Yoshikazu Tanaka, Toyoyuki Ose, Isamu Matsumoto, Koji Kato, Yao Min, Taro Tachibana, Masaharu Sato, Kentaro Naruchi, Fayna Garcia Martin, Hiroshi Hinou, Shin Ichiro Nishimura

Chemical Science 11 ( 19 ) 4999 - 5006 2020.5

Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry (RSC)

© The Royal Society of Chemistry 2020. Aberrantly truncated immatureO-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immatureO-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of “dynamic neoepitopes” elaborated by disease-specificO-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.

DOI： 10.1039/d0sc00317d

Structural basis for assembly and function of a diatom photosystem I-light-harvesting supercomplex. Reviewed International journal

Ryo Nagao, Koji Kato, Kentaro Ifuku, Takehiro Suzuki, Minoru Kumazawa, Ikuo Uchiyama, Yasuhiro Kashino, Naoshi Dohmae, Seiji Akimoto, Jian-Ren Shen, Naoyuki Miyazaki, Fusamichi Akita

Nature communications 11 ( 1 ) 2481 - 2481 2020.5

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

Photosynthetic light-harvesting complexes (LHCs) play a pivotal role in collecting solar energy for photochemical reactions in photosynthesis. One of the major LHCs are fucoxanthin chlorophyll a/c-binding proteins (FCPs) present in diatoms, a group of organisms having important contribution to the global carbon cycle. Here, we report a 2.40-Å resolution structure of the diatom photosystem I (PSI)-FCPI supercomplex by cryo-electron microscopy. The supercomplex is composed of 16 different FCPI subunits surrounding a monomeric PSI core. Each FCPI subunit showed different protein structures with different pigment contents and binding sites, and they form a complicated pigment-protein network together with the PSI core to harvest and transfer the light energy efficiently. In addition, two unique, previously unidentified subunits were found in the PSI core. The structure provides numerous insights into not only the light-harvesting strategy in diatom PSI-FCPI but also evolutionary dynamics of light harvesters among oxyphototrophs.

DOI： 10.1038/s41467-020-16324-3

Structure of a cyanobacterial photosystem I surrounded by octadecameric IsiA antenna proteins. Reviewed International journal

Fusamichi Akita, Ryo Nagao, Koji Kato, Yoshiki Nakajima, Makio Yokono, Yoshifumi Ueno, Takehiro Suzuki, Naoshi Dohmae, Jian-Ren Shen, Seiji Akimoto, Naoyuki Miyazaki

Communications biology 3 ( 1 ) 232 - 232 2020.5

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

Iron-stress induced protein A (IsiA) is a chlorophyll-binding membrane-spanning protein in photosynthetic prokaryote cyanobacteria, and is associated with photosystem I (PSI) trimer cores, but its structural and functional significance in light harvesting remains unclear. Here we report a 2.7-Å resolution cryo-electron microscopic structure of a supercomplex between PSI core trimer and IsiA from a thermophilic cyanobacterium Thermosynechococcus vulcanus. The structure showed that 18 IsiA subunits form a closed ring surrounding a PSI trimer core. Detailed arrangement of pigments within the supercomplex, as well as molecular interactions between PSI and IsiA and among IsiAs, were resolved. Time-resolved fluorescence spectra of the PSI-IsiA supercomplex showed clear excitation-energy transfer from IsiA to PSI, strongly indicating that IsiA functions as an energy donor, but not an energy quencher, in the supercomplex. These structural and spectroscopic findings provide important insights into the excitation-energy-transfer and subunit assembly mechanisms in the PSI-IsiA supercomplex.

DOI： 10.1038/s42003-020-0949-6

Crystal structures clarify cofactor binding of plant tyrosine decarboxylase. Reviewed International journal

Hang Wang, Jian Yu, Yasuharu Satoh, Yusuke Nakagawa, Ryusuke Tanaka, Koji Kato, Min Yao

Biochemical and biophysical research communications 523 ( 2 ) 500 - 505 2020.3

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

Plant tyrosine decarboxylase (TyrDC) is a group II pyridoxal 5'-phosphate (PLP)-dependent decarboxylase that mainly catalyzes the decarboxylation of tyrosine to tyramine. This is biologically important for diverting essential primary metabolites into secondary metabolic pathways. Intensive studies have characterized the effective of PLP-binding and the substrate specificity of mammalian 3,4-dihydroxyphenyl-l-alanine (Dopa) decarboxylases, a member of group II PLP-dependent decarboxylase. However, the characteristics of PLP binding and substrate specificity of plant TyrDCs remain unknown. In this study, we focus on the PLP binding manner, and determined the crystal structures of the apo and PLP binding form of type II TyrDC from Papaver somniferum (PsTyrDCII and PsTyrDCII-PLP). The structures showed that, unlike mammalian Dopa decarboxylase, the binding of PLP does not induce distinct conformational changes of PsTyrDCII regarding the overall structure, but the PLP binding pocket displays conformational changes at Phe124, His203 and Thr262. Combining structural comparation and the obtained biochemical findings, it is demonstrated that PsTyrDCII does not binds PLP tightly. Such characteristics of PLP binding may be required by its catalytic reaction and substrate binding. The activity of TyrDC probably regulated by the concentration of PLP in cells.

DOI： 10.1016/j.bbrc.2019.12.077

Structural basis for the adaptation and function of chlorophyll f in photosystem I. Reviewed International journal

Koji Kato, Toshiyuki Shinoda, Ryo Nagao, Seiji Akimoto, Takehiro Suzuki, Naoshi Dohmae, Min Chen, Suleyman I Allakhverdiev, Jian-Ren Shen, Fusamichi Akita, Naoyuki Miyazaki, Tatsuya Tomo

Nature communications 11 ( 1 ) 238 - 238 2020.1

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

Chlorophylls (Chl) play pivotal roles in energy capture, transfer and charge separation in photosynthesis. Among Chls functioning in oxygenic photosynthesis, Chl f is the most red-shifted type first found in a cyanobacterium Halomicronema hongdechloris. The location and function of Chl f in photosystems are not clear. Here we analyzed the high-resolution structures of photosystem I (PSI) core from H. hongdechloris grown under white or far-red light by cryo-electron microscopy. The structure showed that, far-red PSI binds 83 Chl a and 7 Chl f, and Chl f are associated at the periphery of PSI but not in the electron transfer chain. The appearance of Chl f is well correlated with the expression of PSI genes induced under far-red light. These results indicate that Chl f functions to harvest the far-red light and enhance uphill energy transfer, and changes in the gene sequences are essential for the binding of Chl f.

DOI： 10.1038/s41467-019-13898-5

Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing. Reviewed International journal

Xiaomei Sun, Yuxin Ye, Naofumi Sakurai, Koji Kato, Keizo Yuasa, Akihiko Tsuji, Min Yao

Acta crystallographica. Section F, Structural biology communications 76 ( Pt 1 ) 20 - 24 2020.1

Language：English

Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects β-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48 Å using wavelengths of 1.0 and 2.1 Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.

DOI： 10.1107/S2053230X19016716

Structure of a cyanobacterial photosystem I tetramer revealed by cryo-electron microscopy. Reviewed International journal

Koji Kato, Ryo Nagao, Tian-Yi Jiang, Yoshifumi Ueno, Makio Yokono, Siu Kit Chan, Mai Watanabe, Masahiko Ikeuchi, Jian-Ren Shen, Seiji Akimoto, Naoyuki Miyazaki, Fusamichi Akita

Nature communications 10 ( 1 ) 4929 - 4929 2019.10

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

Photosystem I (PSI) functions to harvest light energy for conversion into chemical energy. The organisation of PSI is variable depending on the species of organism. Here we report the structure of a tetrameric PSI core isolated from a cyanobacterium, Anabaena sp. PCC 7120, analysed by single-particle cryo-electron microscopy (cryo-EM) at 3.3 Å resolution. The PSI tetramer has a C2 symmetry and is organised in a dimer of dimers form. The structure reveals interactions at the dimer-dimer interface and the existence of characteristic pigment orientations and inter-pigment distances within the dimer units that are important for unique excitation energy transfer. In particular, characteristic residues of PsaL are identified to be responsible for the formation of the tetramer. Time-resolved fluorescence analyses showed that the PSI tetramer has an enhanced excitation-energy quenching. These structural and spectroscopic findings provide insights into the physiological significance of the PSI tetramer and evolutionary changes of the PSI organisations.

DOI： 10.1038/s41467-019-12942-8

The pH-dependent conformational change of eukaryotic translation initiation factor 5: Insights into partner-binding manner. Reviewed

Ye Y, Chen M, Kato K, Yao M

Biochemical and biophysical research communications 519 ( 1 ) 186 - 191 2019.10

ErbB3の糖鎖による物性制御メカニズム

高橋素子, 加藤公児, 藤谷直樹, 斎藤充史, 和田芳直, 姚閔

日本生化学会大会プログラム・講演要旨集 92回 [1T10m - 01] 2019.9

Structural basis for energy harvesting and dissipation in a diatom PSII–FCPII supercomplex Reviewed

Ryo Nagao, Koji Kato, Takehiro Suzuki, Kentaro Ifuku, Ikuo Uchiyama, Yasuhiro Kashino, Naoshi Dohmae, Seiji Akimoto, Jian-Ren Shen, Naoyuki Miyazaki, Fusamichi Akita

Nature Plants 5 ( 8 ) 890 - 901 2019.8

Neutron crystallographic study of heterotrimeric glutamine amidotransferase CAB. Reviewed

Li L, Adachi M, Yu J, Kato K, Shinoda A, Ostermann A, Schrader TE, Ose T, Yao M

Acta crystallographica. Section F, Structural biology communications 75 ( Pt 3 ) 193 - 196 2019.3

Function and structure of GH13_31 α-glucosidase with high α-(1→4)-glucosidic linkage specificity and transglucosylation activity. Reviewed

Auiewiriyanukul W, Saburi W, Kato K, Yao M, Mori H

FEBS letters 2018.6

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

Elucidation of the function based on the whole structure of rat liver vault, the largest ribonucleo-protein particle Reviewed

Hideaki Tanaka, Koji Kato, Tomoyuki Sumizawa, Eiki Yamashita

Seikagaku 83 ( 5 ) 392 - 395 2011

2SJ-05 X-ray structure of the vault, the largest cytoplasmic ribonucleo protein particle(2SJ New developments in protein complex research: From molecules to supramolecules and aggregates,The 49th Annual Meeting of the Biophysical Society of Japan)

Tanaka Hideaki, Kato Koji, Yamashita Eiki, Sumizawa Tomoyuki, Zhou Yong, Yao Min, Iwasaki Kenji, Yoshimura Masato, Tsukihara Tomitake

Seibutsu Butsuri 51 S23 2011

ラット肝由来の巨大なRNAタンパク質複合体vaultのX線結晶構造

住澤知之, 加藤公児, 田中秀明

J UOEH Univ Occup Environ Health 32 ( 1 ) 113 2010.3

25.ラット肝由来の巨大なRNAタンパク質複合体vaultのX線結晶構造(第27回産業医科大学学会総会学術講演会記録)

住澤知之, 加藤公児, 田中秀明

産業医科大学雑誌 32 ( 1 ) 2010.3

X-ray structure analysis of a 10MDa biological macromolecule

YAMASHITA Eiki, KATO Koji, TANAKA Hideaki

22 ( 6 ) 284 - 291 2009.11

ラット肝臓由来ボルトの3.5Å分解能での構造解析 Invited

加藤公児, 田中秀明, 月原冨武

2009.10

[X-ray structure of vault: the largest ribonucleoprotein complex]. Reviewed

Kato K, Tanaka H

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 54 1159 - 1165 2009.7

X-ray Crystal Structure of the Vault, Largest Ribonucleoprotein Particle, with a Molecular Weight of 10MDa

TANAKA Hideaki, KATO Koji, YAMASHITA Eiki

Nihon Kessho Gakkaishi 51 ( 3 ) 189 - 194 2009.6

Language：Japanese Publisher：The Crystallographic Society of Japan

Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Although roles in multidrug resistance and innate immunity have been suggested, the cellular function remains unclear. We have determined the X-ray structure of rat liver vault at 3.5 Å resolution. A vault particle shell was composed of 78 MVP (Major vault protein) chains with 39-fold dihedral symmetry. The shoulder domain of MVP is structurally similar to SPFH (stomatin/prohibitin/flotillin/HflK/C) domain involved in lipid raft association.

DOI： 10.5940/jcrsj.51.189

X-ray crystal structural determination of rat vault, a large nucleoprotein complex at 3.5Å resolution. Invited

H. Tanaka, K. Kato, E. Yamashita, T. Sumizawa, Y. Zhou, M. Yao, K. Iwasaki, M. Yoshimura, T. Tsukihara

2009.3

The Structure of Rat Liver Vault at 3.5 Angstrom Resolution Reviewed

Hideaki Tanaka, Koji Kato, Eiki Yamashita, Tomoyuki Sumizawa, Yong Zhou, Min Yao, Kenji Iwasaki, Masato Yoshimura, Tomitake Tsukihara

SCIENCE 323 ( 5912 ) 384 - 388 2009.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC ADVANCEMENT SCIENCE

Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Roles in multidrug resistance and innate immunity have been suggested, but the cellular function remains unclear. We have determined the x-ray structure of rat liver vault at 3.5 angstrom resolution and show that the cage structure consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The shoulder domain is structurally similar to a core domain of stomatin, a lipid-raft component in erythrocytes and epithelial cells.

DOI： 10.1126/science.1164975

The Structure of Rat Liver Vault at 3.5 Angstrom Resolution Reviewed

Hideaki Tanaka, Koji Kato, Eiki Yamashita, Tomoyuki Sumizawa, Yong Zhou, Min Yao, Kenji Iwasaki, Masato Yoshimura, Tomitake Tsukihara

Science 49 ( Supplement 1 ) 2009.1

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas reuoluta) seeds Reviewed

Seiya Yokoyama, Kouji Kato, Atsuko Koba, Yuji Minami, Keiichi Watanabec, Fumio Yagi

PEPTIDES 29 ( 12 ) 2110 - 2117 2008.12

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

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 mu g/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2008.08.007

X-ray structure of the vault purified from rat liver Invited

田中秀明, 加藤公児, 住澤知之, 山下栄樹, 吉村政人, 周勇, 姚閔, 岩崎憲治, 月原冨武

2008.12

A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry Reviewed

Koji Kato, Hideaki Tanaka, Tomoyuki Sumizawa, Masato Yoshimura, Eiki Yamashita, Kenji Iwasaki, Tomitake Tsukihara

ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 64 525 - 531 2008.5

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

Vault is a 12.9 MDa ribonucleoprotein particle with a barrel-like shape, two protruding caps and an invaginated waist structure that is highly conserved in a wide variety of eukaryotes. Multimerization of the major vault protein (MVP) is sufficient to assemble the entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1), as well as a small vault RNA (vRNA), are also associated with vault. Here, the crystallization of vault particles is reported. The crystals belong to space group C2, with unit-cell parameters a = 708.0, b = 385.0, c = 602.9 angstrom, beta = 124.8 degrees. Rotational symmetry searches based on the R factor and correlation coefficient from noncrystallographic symmetry (NCS) averaging indicated that the particle has 39-fold dihedral symmetry.

DOI： 10.1107/S0907444908004277

1P-002 X-ray structure of the vault purified from rat liver(Invited Talk for Early Research in Biophysics Award,Early Research in Biophysics Award)(The 46th Annual Meeting of the Biophysical Society of Japan)

Tanaka Hideaki, Kato Koji, Sumizawa Tomoyuki, Yamashita Eiki, Yoshimura Masato, Yong Zhou, Yao Min, Iwasaki Kenji, Tsukihara Tomitake

Seibutsu Butsuri 48 S21 2008

2P041 X-ray crystal structural analysis of vault from rat liver(29. Protein structure and dynamics (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

TANAKA Hideaki, KATO Koji, Yamashita Eiki, FUJITA Chizuko, SUMIZAWA Tomoyuki, TSUKIHARA Tomitake

Seibutsu Butsuri 46 ( 2 ) S306 2006

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