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))

Toward Biorefinery through the Development of Biomass and Biomolecules Based on Structural Biology

Structural Energy Bioscience Research Section


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. For example, recently we have successfully developed the way to monitor the base conversion reaction by anti-HIV enzyme, A3G protein, in real-time by NMR for the first time. This new method has provided critical information on how this enzyme makes the catalytic action on DNA. Currently, we are developing the way to extract energy and valuable materials that can be used as starting materials of various products from wood biomass. Thus, we pursue to contribute to the paradigm shift from oil refinery to biorefinery. (Prof. Masato KATAHIRA, Assoc. Prof. Takashi NAGATA, Assistant Prof. Tsukasa MASHIMA)


Structure of A3G protein which possesses the anti-HIV activity and the interaction sites (colored) with target virus DNA (upper left). Real-time monitoring of the base-conversion reaction through deamination by A3G with NMR signals (lower left and right).


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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 2011.

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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. Construction of a kinetic model to explain the location-dependent deamination frequency of DNA by anti-HIV protein, APOBEC3G

be utilized to effectively deaminate and destroy the minus strand DNA of HIV. We developed the way to monitor deamination of a cytidine by anti-HIV protein, APOBEC3G (A3G), in real-time using NMR signals. Then, we have found that a cytidine located closer to the 5’ end of single-stranded DNA (ssDNA) is deaminated more frequently that that located less close to the 5’ end. Here, we have constructed a kinetic model that can successfully explain the location-dependent deamination. In this model, A3G slides on ssDNA to either 5’ or 3’ direction with the equal speed, but a catalytic rate of the deamination is larger when A3G approaches to a substrate cytidine in 3’ to 5’ directionality than that in 5’ to 3’ directionality (Figure 2). This difference in the catalytic rate is rationalized from the inspection of the three-dimensional structure of A3G. In this model, the cytidine located closer to the 5’ end has more chance to meet A3G approaching in the 3’ to 5’ directionality, which would result in higher frequency of the deamination for that cytidine. Thus, the experimental result can be successfully explained by this kinetic model. This behavior of A3G is supposed to


Figure 2 The kinetic model constructed to explain the location-dependent deamination frequency along DNA by anti-HIV protein, A3G.

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4. 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 3 The way how dimeric RNA aptamer traps full-length prion protein.

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5. Development of intelligent enzymes that switch their activity in response to K+

The structure of r(GGAGGAGGAGGA) (R12) changes from a single-stranded form to a compact quadruplex one in response to K+. In a hammerhead ribozyme, two portions of the catalytic core are linked with the stem and are located closely to exert the activity. Here, this stem was replaced by R12 (or R11 that lacks the terminal A residue) with some linker residues (Figure 4). One of the newly con-structed ribozymes exhibited enhanced activity in response to K+. It is suggested that the quadruplex formation restored the active catalytic core. Other ri-bozymes exhibited repressed activity in response to K+. It is suggested that the quadruplex prevents the formation of the active core. Thus, we have suc-ceeded in developing intelligent ribozymes that switch their activity either positively or negatively in response to K+. This switching capability may have therapeutic applications because the intra- and extracellular K+concentrations are very different.


Figure 4 Architecture of intelligent ribozymes that switch their activity in response to K+.


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6. Development of Highly Efficient Bioethanol Production Yeast Using Protein Engineering

In this fiscal year, the more efficient xylose fer-mentation and the decrease of xylitol excretion was observed by introducing the strictly NADPH de-pendent XR with the strictly NADP+ dependent XDH. These effects are probably due to the full recycling of coenzymes between the mutated XR and XDH.

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7. Analysis of the structure-function relationships on wood degrading enzymes for better utilization of woody biomass


Cellulose, hemicellulose, and lignin are the major components of wood. Cellulose has been used as a starting material to produce bioethanol and bioplastic. Other two components are also made of chemical structural units, which have potentials to be converted into bioethanol, biomaterials and medicine. However, the complexity of their structure in woody tissue hinders their isolation, as well as characterization of structure and function. To isolate lignin and hemicellulose in the native form so as to be able to use them as starting materials, we have begun to investigate the potentials of protein enzymes that are expressed in fungi that degrade wood. We have subcloned single genes of each cellulase and manganese peroxidase (MnP) of white-rot fungus (highly selective lignin-degrading fungus) that are thought to play major roles in lignin degradation. We have then succeeded to express cellulase in P. pastoris system and MnP in E. coli system. Optimization of the preparation procedure is underway. The further study is in progress to measure their activity, search for cofactors that enhance the activity, and solve the structure.

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Staffs

Professor  Masato Katahira, Email: katahira@@iae.kyoto-u.ac.jp

Associate professor             Takashi Nagata

Assistant professor               Tsukasa Mashima

Postdoctoral fellow              Keiko Kondo

Secretary                             Rika Hamada

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Graduate students

D3  Keisuke Kamba
D3  Yudai Yamaoki
D3  Huyen Nguyen
D3  Lin Meng-I
D2  Mamiko Iida
D2  Wan Li
D1  Wan Hasnida WAN OSMAN
M2  Ayaka Kiyoishi
M2  Kenshi Takagi
M2  Kazuma Nagata
M1  Yuki Ueyama
M1  Shunsuke Ozawa
M1  Ryosuke Fushitani
M1  Yushiro Kose
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