Katahira Lab., Structural Energy Bioscience Research Section
Institute of Advanced Energy, Kyoto University (K-12)

(Bioenergy Research Section, Department of Fundamental Energy Science,                  Graduate School of Energy Science, Kyoto University (K-12))

Structural Energy Bioscience Research Section

1. Introduction

We explore the way how biomolecules such as proteins (involving enzymes) and functional nucleic acids (DNA and RNA) work at atomic resolution based on structural biology with NMR. We determine both static and dynamical structures with the aid of our own development of the new methodology and elucidate the underlying mechanism of functions of these biomolecules. Structural biological approach is also applied to analyze components of wood biomass at atomic resolution. The analysis is usefule to develope the way to extract energy and valuable materials that can be used as starting materials of various products from the wood biomass. Thus, we pursue to contribute to the paradigm shift from oil refinery to biorefinery. Followings are main research achievements in the year of 2018.

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2. Development of the method to analyze components of wood biomass by solution NMR –Analysis of all components of whole wood in an intact state at atomic resolution

We have developed a new way of sample prepa-ration of wood biomass for the analysis by a solution NMR method. This new preparation brings much sharper NMR signals and much better signal to noise ratio. As a result, it is possible to analyze all compo-nents of whole wood biomass in an intact state (no artificial chemical modification) at atomic resolution (Figure 1). We have also developed the method to accurately quantify the components of wood biomass with NMR. We have applied the developed method to monitor the bio-degradation of wood biomass by fungi. Selective degradation of either cellulose or lignin depending on a kind of fungi used was successfully detected. Thus, our NMR method has a wide range of application to identify various components of wood biomass and to monitor their conversion.

Figure 1 1H-13C HSQC spectrum of wood biomass. Identification of cellulose and lignin substructures is illustrated.

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3. Classification and characterization of two new fungal glucuronoyl esterases from Ceriporiopsis subvermispora and Pleurotus eryngii

The lignin-carbohydrate complex (LCC) found in the recalcitrant woody biomass is a complex of lignin and hemicellulose. Fungal glucuronoyl esterases (FGEs) catalyze cleavage of LCC at the ester bond formed between an alcohol group of lignin and the xylan-bound 4-Omethyl-D-glucuronic acid of glucuronoxylans. Here, we built a phylogenetic tree from about 400 putative FGEs obtained on BLAST analysis and defined six main clades. We then found that FGEs of C. subvermispora and P. eryngii belong to clades V and II, respectively, and that FGEs of these clades have not been studied yet. We developed high-yield Pichia pastoris expression systems of the catalytic domains of FGEs of C. subvermispora (CsGE) and P. eryngii (PeGE). We then used benzyl glucuronic acid to confirm the activities of these FGEs. Firstly, we developped the improved protocol to accurately measure the activity. We successfully quantified the hydrolyzed product, glucuronic acid, spectrophotometrically with higher accuracy using our new protocol. It turned out that PeGE exhibits the highest acitivity among FGEs reported so far. Then, we demonstrated that PeGE exhibits high tolerance toward verious denaturing agents, which may make it a potentially more applicable enzyme.

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4. Structural and functional studies of a serine-type glutathione S-transferase of Ceriporiopsis subver-mispora

Glutathione (GSH) S-transferases (GSTs) are en-zymes that catalyze the reactions involved in detox-ification and metabolic pathways. We previously developed high-yield E. coli expression systems of two GSTs of C. subvermispora, CsGST63524 and CsGST83044, and characterized their activities. Here, we solved the crystal structures of the CsGST63524 in ligand-free and GSH-bound forms at 2.45 and 2.50 Å resolutions, respectively. It was found that the res-idue located closest to the sulfur atom of glutathione for hydrogen bonding is Ser21, which indicated that CsGST63524 is a serine-type GST. Functional mu-tagenesis unexpectedly showed that Ser21 is not es-sential for the enzymatic activity, but interestingly revealed that non-canonical amino acid residues, Asn23 and Tyr45, are enzymatically important. We have also identified the putative substrate binding site, which comprises mostly hydrophobic residues, of CsGST63524 (Figure 1). This is reasonable be-cause many of the substrates that CsGST63524 tar-gets are hydrophobic. Additionally, relatively large GSH-bound pocket of CsGST63524 rationally ex-plains its substrate-preference.

Figure 2 Close-up surface representation of the putative substrate-binding site of CsGST63524 located near the GSH-binding site.

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5. Interaction of carbohydrate-binding module from Trichoderma reesei Cel7A with cellohexaose and lignins from Japanese cedar and Eucalyptus glob-ulus

Lignocellulosic biomass, which comprises cellulose, hemicelluloses, and lignin, is the most abundant re-newable carbon resource. The production of bio-based fuels and value-added chemicals from lignocellulosic biomass is attractive, because it has a potential of partial replacement of fossil resources and to overcome environmental issues. Saccharifica-tion of lignocellulosic biomass using cellulolytic enzymes is ideal regarding isolation of the compo-nents intact. However, nonproductive binding of lignin to cellulolytic enzymes has been problematic and need to be avoided. It has been known that car-bohydrate-binding modules (CBMs) of cellulolytic enzymes strongly bind to lignin, but the adsorption mechanism at molecular level is still remained elu-sive. Here, NMR-based identification of binding sites on CBM1 of cellobiohydrolase I (Cel7A) from a hyper-cellulase-producing fungus, Trichoderma reesei, toward cellohexaose and lignins from Japa-nese cedar (C-MWL) and Eucalyptus globulus (E-MWL) was carried out. Firstly, we developed E. coli expression system of TrCBM1, and preparation method to obtain properly folded and stable iso-tope-labeled TrCBM1. Chemical shift perturbation analyses revealed that TrCBM1 adsorbs cellohex-aose highly specifically using two subsites, flat plane surface and cleft, which were located on the opposite side of the protein surface. On the other hand, MWLs, which exhibited higher affinity than cellohexaose toward TrCBM1, were adsorbed using multiple binding sites, including the subsites. Of the residues belonging to the flat plane surface, Gly6 and Gln7 were involved in lignin binding, while Asn29 and Gly34 in cellohexaose binding (Figure 2). It turned out that much larger surface area was used in TrCBM1 to bind with C-MWL than E-MWL. This indicates that the mech-anisms of adsorption toward hardwood and softwood lignins are quite different.

Figure 3 Binding site mapped on the TrCBM1 model structure. (Top) Binding sites of cellohexaose (left), and C- and E-MWLs (right), respectively. (Bottom) Common binding sites of cellohexaose, and C- and E-MWLs.

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6. The mechanism by which the deaminase activity of human APOBEC3F, which depends on the length of single-stranded DNA, is affected by the concentra-tions of APOBEC3F and single-stranded DNA

APOBEC3F (A3F), a member of the human APO-BEC3 (A3) family of single-stranded (ss) DNA cyti-dine deaminases, acts as an anti-human immunodefi-ciency virus 1 (HIV-1) factor by deaminating deox-ycytidines to deoxyuridines in the complementary DNA of the viral genome. Previous studies on A3F were mainly focused on its interaction with an ac-cessory protein of HIV-1 known as a viral infectivity factor, while a full understanding of the deamination behavior of A3F awaits further investigation. Here, we investigated the ssDNA-length-dependence of the deaminase activity of the C-terminal domain (CTD) of A3F. The use of the N214H mutant of A3F-CTD, which exhibits higher deaminase activity than the wild-type, facilitated tracking of the reaction on the real-time NMR time scale. It turned out that A3F-CTD efficiently deaminates the target deox-ycytidine in long ssDNA with a low ssDNA concen-tration ([A3F-CTD] >> [ssDNA]), while the target deoxycytidine in short ssDNA is deaminated effi-ciently by A3F-CTD with a high ssDNA concentra-tion ([A3F-CTD] << [ssDNA]). This property of A3F is quite different from that of the previously studied A3 family member, A3B. In the case of A3B, deami-nation of the target deoxycytidine in short ssDNA is more efficient than that in long ssDNA; the concen-trations of the protein and ssDNA had no effect. This unique property of A3F is rationally interpreted on the basis of its binding characteristics as to ssDNA.

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7. Elucidation of the way how RNA aptamer traps prion protein

We have identified how RNA aptamer binds to and traps a full-length prion protein that causes prion diseases such as mad cow disease. RNA aptamer forms a dimeric architecture and each monomer sim-ultaneously binds to the two sites located in the N-terminal flexible region of the prion protein, re-spectively (Figure 3). Electrostatic and stacking in-teractions contribute to the binding at each site. The tight binding (trapping) inhibits the conversion of prion protein into an abnormal form related to diseases, which would result in repression of prion diseases. Cell-based assay to confirm this idea is in progress.

Figure 4 The way how dimeric RNA aptamer traps full-length prion protein.

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Publications for fiscal year 2018

L. Wan, T. Nagata, M. Katahira,Influence of the DNA sequence/length and pH on deaminase activity, as well as the roles of the amino acid residues around the cata-lytic center of APOBEC3F,Physical Chemistry Chem-ical Physics,20,3109-3117,2018

K. Kamba, T. Nagata, M. Katahira,The C-terminal cytidine deaminase domain of APOBEC3G itself un-dergoes intersegmental transfer for a target search, as revealed by real-time NMR monitoring,Physical Chemistry Chemical Physics,20,2976-2981,2018

Y. Yamaoki, A. Kiyoishi, M. Miyake, F. Kano, M. Mu-rata, T. Nagata, M. Katahira,The first successful ob-servation of in-cell NMR signals of DNA and RNA in living human cells,Physical Chemistry Chemical Physics,20,2982-2985,2018

K. Takada, R. Amano, Y. Nomura, Y. Tanaka, S. Sugiyama, T. Nagata, M. Katahira, Y. Nakamura, T. Kozu, T. Sakamoto,Characterisation of an aptamer against Runt domain of AML1 (RUNX1) by NMR and mutational analyses,FEBS Open Bio,8,264-270,2018

K. Kondo, T. Mashima, T. Oyoshi, R. Yagi, R. Kurokawa, N. Kobayashi, T. Nagata, M. Katahira,Plastic roles of phenylalanine and tyrosine residues of an RGG motif of TLS/FUS in binary and ternary complex formation with the G-quadruplex structures of telomeric DNA and TERRA,Scientific Reports,8,2864,2018

K. Onizuka, A. Usami, Y. Yamaoki, T. Kobayashi, M. Hazemi, T. Chikuni, N. Sato, K. Sasaki, M. Katahira, F. Nagatsugi,Selective alkylation of T-T mismatched DNA using vinyldiaminotriazine-acridine conjugate,Nucleic Acids Research,46,1059-1068,2018

M. Takeda, K. Kondo, S. Sanda, D. Kan, I.K. Borges, I. Suzuki, M. Katahira,Enzymatic degradation of β-1,4-linked N-acetylglucosaminoglucan prepared from Thiothrix nivea,International Journal of Biological Macromolecules,109,323-328,2018

M.I. Lin, T. Nagata, M. Katahira,High yield production of fungal manganase peroxidases by E. coli through soluble expression, and examination of the activities,Protein Expression and Purification,145,45-52,2018

W.H. Wan Osman, M.I. Lin, K. Kondo, T. Nagata, M. Katahira, Characterization of the glutathione S-transferases that belong to the GSTFuA class in Ceri-poriopsis subvermispora: Implications in intracellular detoxification and metabolism of wood-derived com-pounds, Int. J. Biol. Macromolecule., 113, 1158-1166, 2018

A. Kitada, S. Takeoka, K. Kintsu, K. Fukami, M. Sai-mura, T. Nagata, M. Katahira, K. Murase,A Hydronium Solvate Ionic Liquid: Facile Synthesis of Air-Stable Ionic Liquid with Strong Brønsted Acidity,Journal of the Electrochemical Society,165,H121-H127,2018

T. Hayashi, T. Matsuda, T. Nagata, M. Katahira, M. Kinoshita,Mechanism of protein-RNA recognition: Analysis based on statistical mechanics of hydration,Physical Chemistry Chemical Physics,20,14,9167-9180,2018

T. Nagata,Utilizing NMR to study RNA- and com-pound-binding mechanisms of Musashi-1, a stem/progenitor cell marker in various normal and can-cer cells,Integrative Cancer Science and Therapeutics,5,2,2018

H. Nishimura, A. Kamiya, T. Nagata, M. Katahira, T. Watanabe,Direct evidence for α ether linkage between lignin and carbohydrates in wood cell walls,Scientific Reports,8,6538,2018

A. Kitada, K. Kintsu, S. Takeoka, K. Fukami, M. Sai-mura, T. Nagata, M. Katahira, K. Murase,A Hydronium Solvate Ionic Liquid: Ligand Exchange Conduction Driven by Labile Solvation,Journal of The Electro-chemical Society,165,9,H496-H499,2018

Y. Kawasaki, K. Kurosaki, D. Kan, I. K. Borges, A. Sa-take, M. Sato, K. Kondo, M. Katahira, I. Suzuki, M. Takeda,Identification and characterization of the S-layer formed on the sheath of Thiothrix nivea,Archives of microbiology,2018

N. Kobayashi, Y. Hattori, T. Nagata, S. Shinya, P. Gun-tert, C. Kojima, T. Fujiwara,Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks,Bioinformatics,2018

M.I. Lin, A. Hiyama, K. Kondo, T. Nagata, M. Katahira,Classification of fungal glucuronoyl esterases (FGEs), and characterization of two new FGEs from Ceriporiop-sis subvermispora and Pleurotus eryngii,Applied Mi-crobiology and Biotechnology,2018

S.S. Masoud, Y. Yamaoki, Y. Ma, A. Marchand, F.R. Winnerdy, V. Gabelica, A.T. Phan, M. Katahira, K. Na-gasawa,Analysis of interaction between telomeric i-motif DNA and a cyclic tetraoxazole compound,ChemBioChem,19,1-6,2018

Y. Ohara, Y. Ozeki, Y. Tateishi, T. Mashima, F. Arisaka, Y. Tsunaka, Y. Fujiwara, A. Nishiyama, Y. Yoshida, K. Kitadokoro, H. Kobayashi, Y. Kaneko, I. Najagawa, R. Maekura, S. Yamamoto, M. Katahira, S. Matsumoto,Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis,PLoS ONE,13,10,e0204160,2018

Z. Chen, Y. Tonouchi, K. Matsumoto, M. Saimura, R. Atkin, T. Nagata, M. Katahira, R. Hagiwara,Partially Naked Fluoride in Solvate Ionic Liquids,Journal of Physical Chemistry Letters,9,22,6662-6667,2018

M. Katahira, T. Mashima,Introduction to Nucleic Acids NMR,Encyclopedia of Biophysics 2nd Edition,2018

R. Kurokawa, M. Katahira, et al.,Multiplicity in long noncoding RNA in living cells,Biomedical Sciences,4,18-23,2018

T. Tokunaga, T. Nagata, T. Suetomi, S. Oshiro, K. Kondo, M. Katahira, T. Watanabe,NMR Analysis on Molecular Interaction of Lignin with Amino Acid Residues of Carbohydrate-Binding Module from Trichoderma reesei Cel7A,Scientific Reports,9,1,1977,2019

O.W.H. Wan ,B. Mikami, N. Saka, K. Kondo, T. Nagata, M. Katahira,Structure of a serine-type glutathione S-transferase of Ceriporiopsis subvermispora and iden-tification of the enzymatically important non-canonical residues by functional mutagenesi,Biochemical and Biophysical Research Communications,510,1,177-183,2019

M. Iida, T. Mashima, Y. Yamaoki, M. So, T. Nagata, M. Katahira, The anti-prion RNA aptamer R12 disrupts the Alzheimer’s disease-related complex between prion and amyloid β, FEBS J., doi: 10.1111/febs.14819, 2019

W.H. Wan Osman, B. Mikami, N. Saka, K. Kondo, M.I. Lin, T. Nagata, M. Katahira, Identification of key resi-dues for activities of atypical glutathione S-transferase of Ceriporiopsis subvermispora, a selective degrader of lignin in woody biomass, by crystallography and func-tional mutagenesis, Int. J. Biol. Macromol., doi: 10.1016/j.ijbiomac.2019.03.199, 2019


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Professor                              Masato KATAHIRA
                                                (Email: katahira.masato.6u@@kyoto-u.ac.jp)

Associate professor            Takashi NAGATA

Assistant professor             Yudai YAMAOKI

Postdoctoral fellow              Keiko KONDO

Postdoctoral fellow              Keisuke KAMBA

Collaborative researcher     Bunzo MIKAMI

Collaborative researcher     Masahiro KINOSHITA

Collaborative researcher     Tsukasa MASHIMA

Collaborative researcher     Tomohiko HAYASHI

Collaborative researcher     Nesreen Hamad Abdel Gawwad HAMAD

Secretary                               Naomi MURAKAMI

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Graduate students

D3  Mohammed Oksh Mohammed MOUSA
D3  Mohammed Mahmoud Afaf Sobhi ELADL
D2  Wei Hsun TU
D1  Kenneth TEO sze kai
D1  Mohamed Mahmoud Omar Sobhi ELADL
M2  Chihiro NAKAYAMA
M2  Yusei YAGI
M1  Weile DAI
M1  Chenchen JI
M1  Soichiro MATSUO
M1  Taiki MORITA

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