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 pro-teins (involving enzymes) and functional nucleic acids (DNA and RNA) work at atomic resolution based on structural biology with NMR. We deter-mine both static and dynamical structures with the aid of our own development of the new methodology and elucidate the underlying mechanism of func-tions of these biomolecules. Structural biological approach is also applied to analyze enzymes in-volved in degradation of wood biomass at atomic resolution. The analysis is useful to develop 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.

Back to top

2. Activity analysis of PeGE on the cleavage of es-ter-linkage in natural woody biomass

The major components of woody biomass, cellulose, hemicellulose, and lignin, are promising carbon-neutral resources to produce biofuels and various materials. These components form the rigid complex so-called lignin-carbohydrate complex (LCC) in natural biomass. One of the direct linkage contributing to LCC formation is ester-linkage between lignin and glucuronic acid residue in hemicellulose. Fungal glucuronoyl esterases (FGEs) catalyze the cleavage of the ester linkage and thereby facilitate the isolation of woody biomass components. Our previous study revealed that FGE from Pleurotus eryngii (PeGE) has the highest activity toward a model substrate, benzyl glucuronic acid. This year, we have investigated the activity of PeGE on the cleavage of the ester-linkage in natural LCC. NMR spectra of the LCC fraction that was extracted from natural woody biomass were recorded before and after the treatment by PeGE. Change in the signal intensity of the ester-linked lignin and glucuronic acid residue proved that PeGE can catalyze the cleavage of the ester-linkage in woody biomass.

Back to top

3. Structural and functional analysis of lytic poly-saccharide monooxygenase

Saccharification of cellulose by cellulolytic enzymes is a crucial process in the production of 2nd-generation biofuels. Lytic polysaccharide monooxygenases (LPMOs), which catalyze oxidative cleavage of glycosidic linkage, are known to have a synergistic effect on the activity of cellulases on the degradation of crystalline cellulose. Previously we had obtained a crystal of LPMO of a white-rot fungus and solved the crystal structure. We further refined the structure, which revealed that this LPMO has a canonical structure of AA9 family, having a copper ion at the catalytic center (Figure 1). Some polar amino acid and three tyrosine residues around the catalytic center apparently participate in the binding with cellulose. Furthermore, the synergy of LPMO and cellulase cocktail was investigated. In our previous study, simultaneous use of LPMO and cellulase cocktail exerted a 1.3-fold higher glucose yield from crystalline cellulose than the individual use of LPMO and cellulase cocktail. This year we optimized the conditions for the cellulose degradation, by which a 4.7-fold higher glucose yield was recorded.

Figure 1 Over-all structure (a) and copper-coordination (b) of LPMO. The copper ion and its coordinating residues are connected with dashed lines and distances.

Back to top

4. Aggregation of FUS induced by mechanistic shear stress on pipetting and its suppression by non-coding RNA

Fused in sarcoma (FUS) has been considered as a molecular link between apparently different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Although the neurodegenerative diseases have different manifestations, FUS aggregation is associated with all of them, which suggests a common pathway for their neuropathologies. We found that FUS transforms into the amorphous aggregation state as an instant response to the shear stress caused by usual pipetting, by means of fluorescence spectroscopy, fluorescence microscopy, and transmission electron microscopy (TEM) (Figure 2). The more the number of strokes of pipetting is, the more the number of aggregates is. It was also revealed that non-coding RNA can suppress the transformation of FUS into aggregates. There is the possibility that RNA bound to the C-terminal region of FUS masks the interface required for the formation of aggregates, resulting in the prevention of aggregate formation. It is also likely that RNA bound to the C-terminal region of FUS neutralizes the cations and reduces the cation-π interaction between the N- and C-terminal regions, resuktinh in the prevention of aggrergate formation. The suppressive effect of RNA on FUS aggregation is sequence-dependent. These results suggested that the non-coding RNA could be a prospective suppressor of FUS aggregation caused by mechanistic stress in cells. Our finding might serve for the development of therapies for neurodegenerative diseases by using RNA as aggregation inhibitors.

Figure 2 The TEM image showing FUS aggregates formed after 30 strokes of pipetting.

Back to top

5. The detection of the DNA triplex structure in the living human cells

DNAs reportedly fold into not only a double-helix structure but also a triple-helix or triplex structure under in vitro conditions. However, the formation of the triplex structure of DNA in living human cells has not been proven. Here, the oligodeoxynucleotide (triplex ODN) that forms a triplex structure under in vitro conditions was introduced into HeLa cells by cell-resealing system utilizing a pore-forming toxin. The NMR spectrum of this sample was recorded, in which the imino proton signals of the triplex ODN inside the living cells were clearly detected. Most importantly in the living cells, all the imino proton signals that correspond to the triplex structure of the triplex ODN formed under in vitro conditions were commonly observed. This result indicates that the triplex ODN forms the triplex structure that is similar, if not the same, to that formed in in vitro even in the living human cells.

Back to top

6. Interaction of human origin recognition complex subunit 1 with human G-quadruplex DNAs

DNA replication initiates from particular loci in genomes called replication origins. Origin recognition complex (ORC) binds to a replication origin and recruits other replication factors. ORC in some organisms reportedly bind to a replication origin sequence-specifically. However, the manners by which human ORC (hORC) recognizes a replication origin is ellusive. Genome-wide studies revealed previously that guanine (G)-rich sequences are present in most replication origins in human genome. Our previous study implied that the amino acid region 413-511 of the first subunit of hORC, hORC1(413-511), binds preferentially to G-rich DNAs that form a G-quadruplex (G4) structure. Here, we investigated the interaction of hORC1(413-511) with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1(413-511) binds preferentially to DNAs that formed G4 structures over ones having double-stranded structures. CD and NMR clearly showed that the G4 structures of those G-rich DNAs were retained even after complex formation. Importantly, NMR chemical shift perturbation analyses revealed that hORC1(413-511) primarily binds to the external G-tetrad planes of the G4 structures. Our study propose that human ORC1 may recognize replication origins through the G4 structure.

Back to top

Publication lists

  • Original Papers
  • Review Papers

    Back to top


    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

    Back to top

    Graduate students

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

    Back to top