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Article ; Online: Structural and functional insights into the enzymatic activities of lipases from Burkholderia stagnalis and Burkholderia plantarii.

Kataoka, Saori / Kawamoto, Sayuri / Kitagawa, Sayuri / Kugimiya, Wataru / Tsumura, Kazunobu / Akutsu, Yukie / Kubota, Tomomi / Ishikawa, Kazuhiko

FEBS letters

2024

Abstract: Lipases with high interesterification activity are important enzymes for industrial use. The lipase from Burkholderia stagnalis (BsL) exhibits higher interesterification activity than that from Burkholderia plantarii (BpL) despite their significant ... ...

Abstract	Lipases with high interesterification activity are important enzymes for industrial use. The lipase from Burkholderia stagnalis (BsL) exhibits higher interesterification activity than that from Burkholderia plantarii (BpL) despite their significant sequence similarity. In this study, we determined the crystal structure of BsL at 1.40 Å resolution. Utilizing structural insights, we have successfully augmented the interesterification activity of BpL by over twofold. This enhancement was achieved by substituting threonine with serine at position 289 through forming an expansive space in the substrate-binding site. Additionally, we discuss the activity mechanism based on the kinetic parameters. Our study sheds light on the structural determinants of the interesterification activity of lipase.
Language	English
Publishing date	2024-04-24
Publishing country	England
Document type	Journal Article
ZDB-ID	212746-5
ISSN	1873-3468 ; 0014-5793
ISSN (online)	1873-3468
ISSN	0014-5793
DOI	10.1002/1873-3468.14883
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Article ; Online: Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

Kozome, Dan / Uechi, Keiko / Taira, Toki / Fukada, Harumi / Kubota, Tomomi / Ishikawa, Kazuhiko

Applied and environmental microbiology

2022 Volume 88, Issue 12, Page(s) e0065222

Abstract: Chitin is a biopolymer ... ...

Abstract	Chitin is a biopolymer of
MeSH term(s)	Antifungal Agents/chemistry ; Chitin/chemistry ; Chitinases/chemistry ; Disulfides ; Enzyme Stability ; Ficus/enzymology ; Fungi ; Latex ; Proline
Chemical Substances	Antifungal Agents ; Disulfides ; Latex ; Chitin (1398-61-4) ; Proline (9DLQ4CIU6V) ; Chitinases (EC 3.2.1.14)
Language	English
Publishing date	2022-06-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/aem.00652-22
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Article ; Online: Core fucose‐specific Pholiota squarrosa lectin (PhoSL) as a potent broad‐spectrum inhibitor of SARS‐CoV‐2 infection

Yamasaki, Kazuhiko / Adachi, Naruhiko / Ngwe Tun, Mya Myat / Ikeda, Akihito / Moriya, Toshio / Kawasaki, Masato / Yamasaki, Tomoko / Kubota, Tomomi / Nagashima, Izuru / Shimizu, Hiroki / Tateno, Hiroaki / Morita, Kouichi

The FEBS Journal. 2023 Jan., v. 290, no. 2 p.412-427

2023

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein (S protein) is highly N‐glycosylated, and a “glycan shield” is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's ...

Abstract	Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein (S protein) is highly N‐glycosylated, and a “glycan shield” is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N‐glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40‐amino‐acid chemically synthesised peptide specific to core‐fucosylated N‐glycans. The affinities were at a low nanomolar level, which were ~ 1000‐fold stronger than those between PhoSL and the core‐fucosylated N‐glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad‐spectrum effect on SARS‐CoV‐2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL–S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.
Keywords	Pholiota ; Severe acute respiratory syndrome coronavirus 2 ; electron microscopy ; glycosylation ; lectins ; peptides
Language	English
Dates of publication	2023-01
Size	p. 412-427.
Publishing place	John Wiley & Sons, Ltd
Document type	Article ; Online
Note	JOURNAL ARTICLE
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.16599
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Article ; Online: Enhanced affinity of racemic phosphorothioate DNA with transcription factor SATB1 arising from diastereomer-specific hydrogen bonds and hydrophobic contacts.

Yamasaki, Kazuhiko / Akutsu, Yukie / Yamasaki, Tomoko / Miyagishi, Makoto / Kubota, Tomomi

Nucleic acids research

2020 Volume 48, Issue 8, Page(s) 4551–4561

Abstract: Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. ... ...

Abstract	Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. Here, we used isothermal titration calorimetry and X-ray crystallography to investigate the thermodynamic and structural properties of the interaction between the primary DNA-binding domain (CUTr1) of transcription factor SATB1 and dodecamer DNAs with racemic phosphorothioate modifications at the six sites known to contact CUTr1 directly. For both the modified and unmodified DNAs, the binding reactions were enthalpy-driven at a moderate salt concentration (50 mM NaCl), while being entropy-driven at higher salt concentrations with reduced affinities. The phosphorothioate modifications lowered this susceptibility to salt, resulting in a significantly enhanced affinity at a higher salt concentration (200 mM NaCl), although only some DNA molecular species remained interacting with CUTr1. This was explained by unequal populations of the two diastereomers in the crystal structure of the complex of CUTr1 and the phosphorothioate-modified DNA. The preferred diastereomer formed more hydrogen bonds with the oxygen atoms and/or more hydrophobic contacts with the sulfur atoms than the other, revealing the origins of the enhanced affinity.
MeSH term(s)	Crystallography, X-Ray ; DNA/chemistry ; DNA/metabolism ; Hydrogen Bonding ; Hydrophobic and Hydrophilic Interactions ; Matrix Attachment Region Binding Proteins/chemistry ; Matrix Attachment Region Binding Proteins/metabolism ; Models, Molecular ; Phosphorothioate Oligonucleotides/chemistry ; Protein Domains ; Stereoisomerism ; Thermodynamics
Chemical Substances	Matrix Attachment Region Binding Proteins ; Phosphorothioate Oligonucleotides ; SATB1 protein, human ; DNA (9007-49-2)
Language	English
Publishing date	2020-03-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkaa170
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Article: [Crystal structure of polypeptide-GalNAc transferases: lectin domain and catalytic domain].

Kubota, Tomomi

Seikagaku. The Journal of Japanese Biochemical Society

2007 Volume 79, Issue 4, Page(s) 365–370

MeSH term(s)	Animals ; Cattle ; Crystallization ; Humans ; Lectins ; N-Acetylgalactosaminyltransferases/chemistry ; N-Acetylgalactosaminyltransferases/physiology ; Protein Conformation ; Protein Structure, Tertiary ; Rats ; Substrate Specificity ; Polypeptide N-acetylgalactosaminyltransferase
Chemical Substances	Lectins ; N-Acetylgalactosaminyltransferases (EC 2.4.1.-)
Language	Japanese
Publishing date	2007-05-17
Publishing country	Japan
Document type	Journal Article ; Review
ZDB-ID	282319-6
ISSN	0037-1017
ISSN	0037-1017
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Article ; Online: Core fucose-specific Pholiota squarrosa lectin (PhoSL) as a potent broad-spectrum inhibitor of SARS-CoV-2 infection.

The FEBS journal

2022 Volume 290, Issue 2, Page(s) 412–427

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's ...

Abstract	Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.
MeSH term(s)	Humans ; COVID-19 ; Fucose/chemistry ; Lectins/pharmacology ; Polysaccharides/chemistry ; SARS-CoV-2/drug effects ; Antiviral Agents/pharmacology ; Pholiota/chemistry
Chemical Substances	Fucose (28RYY2IV3F) ; Lectins ; Polysaccharides ; Antiviral Agents
Language	English
Publishing date	2022-09-05
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.16599
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Article: Crystal structures of FMN‐bound and FMN‐free forms of dihydroorotate dehydrogenase from Trypanosoma brucei

Kubota, Tomomi / Tani, Osamu / Yamaguchi, Tomohiko / Namatame, Ichiji / Sakashita, Hitoshi / Furukawa, Koji / Yamasaki, Kazuhiko

FEBS Open Bio. 2018 Apr., v. 8, no. 4

2018

Abstract: Dihydroorotate dehydrogenase (DHODH) is a flavin‐binding enzyme essential for pyrimidine biosynthesis, which converts dihydroorotate to orotate. Three‐dimensional structures of cytosolic DHODH of parasitic protozoa are of interest in drug discovery for ... ...

Abstract	Dihydroorotate dehydrogenase (DHODH) is a flavin‐binding enzyme essential for pyrimidine biosynthesis, which converts dihydroorotate to orotate. Three‐dimensional structures of cytosolic DHODH of parasitic protozoa are of interest in drug discovery for neglected tropical diseases, especially because these enzymes possess significantly different structural and functional properties from the membrane‐associated human enzyme. The existing crystal structures of the flavin mononucleotide (FMN)‐bound DHODHs reveal a number of interactions stabilizing FMN. However, to understand the binding mechanism correctly, it is necessary to compare the structures of the FMN‐bound and FMN‐free forms, because the protein moiety of the former is not necessarily the same as the latter. Here, we prepared the FMN‐free DHODH of Trypanosoma brucei using an Escherichia coli overexpression system. Although this apoform lacks enzymatic activity, simple incubation with FMN activated the enzyme. It was stable enough to be crystallized, enabling us to determine its structure by X‐ray crystallography at 1.6 Å resolution. We also determined the FMN‐bound form at 1.8 Å resolution. Although the two structures have essentially the same scaffold, we observed flipping of a peptide‐bond plane in the vicinity of the FMN‐binding site, accompanied by an alternative hydrogen‐bonding pattern. Comparisons of B factors of the protein main chain revealed that binding of FMN decreased flexibility of most of the residues at the FMN‐binding site, but increased flexibility of a lid‐like loop structure over the active center. This increase was ascribed to a conformational change in an FMN‐contacting residue, Asn195, which induced a rearrangement of a hydrogen‐bond network of the residues comprising the lid.
Keywords	Escherichia coli ; Trypanosoma brucei ; X-ray diffraction ; biosynthesis ; drugs ; enzyme activity ; humans ; hydrogen bonding ; moieties ; oxidoreductases
Language	English
Dates of publication	2018-04
Size	p. 680-691.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	2651702-4
ISSN	2211-5463
ISSN	2211-5463
DOI	10.1002/2211-5463.12403
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Article ; Online: Current status of pediatric deceased donor liver transplantation: Lessons learned from a high-volume center in Japan where living donation remains predominant.

Sakamoto, Seisuke / Uchida, Hajime / Shimizu, Seiichi / Yanagi, Yusuke / Takeda, Masahiro / Kubota, Tomomi / Nakazato, Yayoi / Fukuda, Akinari / Kasahara, Mureo

Journal of hepato-biliary-pancreatic sciences

2020 Volume 28, Issue 11, Page(s) 1014–1022

Abstract: Background: In Japan, a recent gradual increase in deceased donor donation has expanded opportunities for pediatric patients to obtain deceased grafts.: Methods: Forty-three children underwent deceased donor liver transplantation (DDLT) at our ... ...

Abstract	Background: In Japan, a recent gradual increase in deceased donor donation has expanded opportunities for pediatric patients to obtain deceased grafts. Methods: Forty-three children underwent deceased donor liver transplantation (DDLT) at our institute before February 2020. Twenty-five patients received a split or reduced graft and 18 patients received a whole graft. The clinical outcomes of DDLT were retrospectively analyzed. Results: The main organ resource was split/reduced grafts retrieved from adult donors; however, the number of whole grafts retrieved from pediatric donors has increased. The rates of major complications were similar in the two groups. The 5-year graft survival rate of patients who received a split/reduced graft (78.0%) was lower than that of patients who received a whole graft (88.9%; P = .40). The 3-year graft survival rates of patients who recently received a split/reduced graft and a whole graft improved to 92.3% and 91.7%, respectively. Conclusions: The recent amendment of the organ allocation system, especially the introduction of pediatric prioritization, can effectively increase the chance to obtain deceased donor grafts for pediatric DDLT in Japan. The recent refinements in donor and recipient selection and in the surgical technique of split DDLT can improve the outcomes of pediatric DDLT in Japan.
MeSH term(s)	Adult ; Child ; Graft Survival ; Humans ; Japan ; Liver Transplantation ; Living Donors ; Retrospective Studies ; Treatment Outcome
Language	English
Publishing date	2020-12-30
Publishing country	Japan
Document type	Journal Article
ZDB-ID	2536236-7
ISSN	1868-6982 ; 1868-6974
ISSN (online)	1868-6982
ISSN	1868-6974
DOI	10.1002/jhbp.886
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Article ; Online: Age and CD20 Expression Are Significant Prognostic Factors in Human Herpes Virus-8-negative Effusion-based Lymphoma.

Kubota, Tomomi / Sasaki, Yosuke / Shiozawa, Eisuke / Takimoto, Masafumi / Hishima, Tsunekazu / Chong, Ja-Mun

The American journal of surgical pathology

2018 Volume 42, Issue 12, Page(s) 1607–1616

Abstract: Human herpes virus-8 (HHV-8)-negative effusion-based lymphoma (HHV-8-negative EBL) can be distinguished from primary effusion lymphoma based on clinical and pathologic findings. Although the morphology between the 2 is similar and they both originate ... ...

Abstract	Human herpes virus-8 (HHV-8)-negative effusion-based lymphoma (HHV-8-negative EBL) can be distinguished from primary effusion lymphoma based on clinical and pathologic findings. Although the morphology between the 2 is similar and they both originate from body cavities with serous effusions and are characterized by lack of tumor masses, HHV-8-negative EBL generally occurs in older patients, and has favorable response to therapy and better prognosis than primary effusion lymphoma. However, no systematic studies have investigated prognostic factors in patients with HHV-8-negative EBL. In this report, clinical and pathologic characteristics of 67 cases of HHV-8-negative EBL, including 2 of our own cases, were analyzed. Univariate analyses revealed older age (70 y and above), Japanese ethnicity, pericardial effusion, CD20 expression, and chemotherapy with rituximab were significantly favorable prognostic factors. Peritoneal effusion was identified as an unfavorable prognostic factor. In the multivariate analysis, age and CD20 expression were independent prognostic factors (P=0.013 and 0.003, respectively). A past history of induced fluid overload, hepatitis C viral infection, and peritoneal effusion were significantly correlated with patients aged below 70 years, while pericardial and pleural effusions were significantly correlated with patients aged 70 years and above. A comparison of cases with and without CD20 expression revealed that Japanese ethnicity and pericardial effusion were significantly correlated with CD20 expression, whereas a past history of induced fluid overload and peritoneal effusion were significantly correlated with the absence of CD20. We concluded that older age and CD20 expression are significant and favorable independent prognostic factors of HHV-8-negative EBL.
MeSH term(s)	Adult ; Age Factors ; Aged ; Aged, 80 and over ; Antigens, CD20/analysis ; Antineoplastic Agents, Immunological/therapeutic use ; Asian Continental Ancestry Group ; Biomarkers, Tumor/analysis ; Fatal Outcome ; Female ; Herpesvirus 8, Human/isolation & purification ; Humans ; Japan ; Lymphoma, Primary Effusion/drug therapy ; Lymphoma, Primary Effusion/ethnology ; Lymphoma, Primary Effusion/immunology ; Lymphoma, Primary Effusion/virology ; Male ; Middle Aged ; Risk Factors ; Rituximab/therapeutic use ; Time Factors ; Treatment Outcome
Chemical Substances	Antigens, CD20 ; Antineoplastic Agents, Immunological ; Biomarkers, Tumor ; Rituximab (4F4X42SYQ6)
Language	English
Publishing date	2018-09-27
Publishing country	United States
Document type	Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	752964-8
ISSN	1532-0979 ; 0147-5185
ISSN (online)	1532-0979
ISSN	0147-5185
DOI	10.1097/PAS.0000000000001168
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Article: Crystal structures of FMN-bound and FMN-free forms of dihydroorotate dehydrogenase from

Kubota, Tomomi / Tani, Osamu / Yamaguchi, Tomohiko / Namatame, Ichiji / Sakashita, Hitoshi / Furukawa, Koji / Yamasaki, Kazuhiko

FEBS open bio

2018 Volume 8, Issue 4, Page(s) 680–691

Abstract: Dihydroorotate dehydrogenase (DHODH) is a flavin-binding enzyme essential for pyrimidine biosynthesis, which converts dihydroorotate to orotate. Three-dimensional structures of cytosolic DHODH of parasitic protozoa are of interest in drug discovery for ... ...

Abstract	Dihydroorotate dehydrogenase (DHODH) is a flavin-binding enzyme essential for pyrimidine biosynthesis, which converts dihydroorotate to orotate. Three-dimensional structures of cytosolic DHODH of parasitic protozoa are of interest in drug discovery for neglected tropical diseases, especially because these enzymes possess significantly different structural and functional properties from the membrane-associated human enzyme. The existing crystal structures of the flavin mononucleotide (FMN)-bound DHODHs reveal a number of interactions stabilizing FMN. However, to understand the binding mechanism correctly, it is necessary to compare the structures of the FMN-bound and FMN-free forms, because the protein moiety of the former is not necessarily the same as the latter. Here, we prepared the FMN-free DHODH of
Language	English
Publishing date	2018
Publishing country	England
Document type	Journal Article
ISSN	2211-5463
ISSN	2211-5463
DOI	10.1002/2211-5463.12403
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