Discovering novel enzymes elucidates unknown intracellular activities.

Profile

Toshihisa Kotake

Associate Professor, Plant glycobiology, Area of Biochemistry and Molecular Biology, Division of Life Science, Graduate School of Science and Engineering

 

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Career

March 1995	Graduation from the Faculty of Integrated Arts and Sciences, Hiroshima University
Between April 1997 and March 2000	Research fellow (DC) of the Japan Society for the Promotion of Science
March 2000	Received PhD degree from the Graduate School of Biosphere Science, Hiroshima University
Between April and December 2000	Postdoctoral fellow at the Research Institute for Biological Sciences, Okayama
Between January and June 2001	Research fellow of the Japan Science and Technology Corporation
July 2001~	Associate professor after the period of assistant and assistant professor at the Graduate School of Science and Engineering, Saitama University

Awards

• JSPP Plant and Cell Physiology Award for the Paper of Excellence (2005)
• Young Researcher Award from the GlycoTOKYO (2009)
• Botanical Society Award for Young Scientists from the Botanical Society of Japan (2010)

 

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 Discovering Novel Enzymes Elucidates Unknown Intracellular Activities.

– Elucidate the Unresolved Phenomena of the Plant Cell Wall –

What is present in the plant cell, but is absent in animal cells? What comes to mind first may be the cell wall or chloroplast. The cell wall determines the shapes and sizes of plant cells. Besides the chloroplasts, the cell wall characterizes plants. In fact, the cell wall supports the bodies of huge trees, produce fluffy cotton, and provide crisp texture to fruits. Since ancient times, human beings have utilized the cell wall for fuels (e.g., firewood), clothes (e.g., hemp and cotton), foods, and paper. How is the cell wall constructed? To address this question, we have focused on sugar nucleotides.

The cell wall is mainly composed of polysaccharides, such as cellulose, hemicellulose, and pectin. Sugar nucleotides are raw materials for the synthesis of these polysaccharides. For example, cellulose and xylan are synthesized from UDP-glucose and -xylose, respectively. We recently discovered novel enzymes for the synthesis of various sugar nucleotides. The existence of novel enzymes suggests the existence of unknown chemical reactions (enzymes are proteins that catalyze chemical reactions). Plants with cell walls should have developed a mechanism to synthesize sugar nucleotides during evolution. Currently, we are also developing plants suitable for biofuel, plants suitable for forcing culture, and plants resistant to environmental stress.

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Process

Discovery of novel enzymes

1. Novel enzymes
   The first step is to establish a method for measuring the activities of novel enzymes, because chemical reactions are catalyzed by enzymes. If activity of a certain novel enzyme is confirmed in a plant, the enzyme should exist in the plant.
2. Bulk purchase of vegetables at market
   Bulk purchase of excellent plant materials is also important for research. We usually use a few kilograms, and sometimes several dozens of kilograms. Recently, we often use sprouting peas (pea sprout: votive candle) and radishes.
3. Purification by chromatography
   A sample is repeatedly passed through a resin-packed column (chromatography) to remove contaminants and purify a novel enzyme. Recently, fewer researchers can conduct this procedure than before. Thus, chromatography has become a specialized technique.
4. Purification of a gene
   First, the amino acid sequence of a purified enzyme is determined. This step can identify the enzyme to some extent. Subsequently, a relevant gene is isolated based on the amino acid sequence. cDNA is synthesized from RNA extracted from a plant. The gene of interest is PCR-amplified for recovery.
5. Preparation of a recombinant protein
   The isolated gene is expressed in Escherichia coli or yeast to produce a large amount of a novel enzyme. The enzyme is investigated regarding their reaction conditions and novel activities.

 

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Research on the Synthesis and Metabolism of Sugar Nucleotides

 

Finally, carbons assimilated by photosynthesis mostly turn into cell wall polysaccharides through sugar nucleotides.

Plants absorb carbon dioxide through photosynthesis. The carbon dioxide mostly turns into sugar nucleotides within the cell to be used for the synthesis of cell wall polysaccharides (e.g., cellulose, hemicellulose, and pectin).
Artificially regulating the synthesis and metabolism of sugar nucleotides may increase the synthesis of cell wall polysaccharides by plants.

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Flow of carbon assimilation and synthetic pathway of sugar nucleotides

The synthesis of cell wall polysaccharides is controlled at the level of sugar nucleotides and by glycosyltransferase activity.

• Sugar nucleotides are “raw materials” for cell wall polysaccharides
• Glycosyltransferase activity and sugar nucleotide level are critical.

Sugar nucleotides are raw materials for cell wall polysaccharides Plant cell walls contain various polysaccharides, such as xyloglucan, a kind of hemicellulose, and glucuronoxylan, as well as cellulose. All of them are synthesized from certain sugar nucleotides. Activating or suppressing the synthesis and metabolism of sugar nucleotides can increase or decrease the accumulation of polysaccharide

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Flow of carbon assimilation and synthetic pathway of sugar nucleotides

The strength of the stem (internode) of Kamairazu, a rice mutant, is so reduced that the stem is torn off easily when bent.

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Kamairazu mutant with the strength of the stem significantly reduced

Electron micrographs: Collaboration with Professor Yasuko Kaneko of the Faculty of Education

We discovered a novel enzyme producing sugar nucleotides, UDP-sugar pyrophosphorylase.

This enzyme was given an enzyme number EC2.7.7.64 by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology.

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Kamairazu mutant of rice, which breaks easily

Kamairazu mutant of rice, as the name implies (no need for sickle), is a mutant that is broken and torn off easily by hand. This mutant has a thin cell wall because of the abnormal synthesis and construction of the cell wall.

If the phenomena of Kamairazu mutant can be reversed, plants that can synthesize larger amounts of cell wall polysaccharides from carbon dioxide can be created.

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Mapping-based cloning of a causative gene

• In this analysis, the Bc6 gene was narrowed down to the 55-58-cM region of chromosome 9.
• Top, chromosome 9: Middle, BAC clones and DNA markers: Bottom, Schematic diagram of Bc6 gene
• A mutated gene was narrowed down to the region underlined in red by mapping.

The difference in the gene sequences between japonica and indica cultivars of rice was utilized to identify a causative locus (address). Kamairazu mutant (Bc6) was found to have an abnormal catalytic subunit of cellulose synthase.

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Arabinogalactan protein, a cell wall glycoprotein

Arabinogalactan protein (AGP) is a signaling molecule that functions outside of the cell. AGP is involved in various phenomena, such as the growth, differentiation, reproduction, and stress tolerance of plants. We utilize a degrading enzyme that acts specifically on the carbohydrate moiety of AGP to elucidate the functions of AGP.
Subjects of AGP research

• Sugar chains are important for the functions of AGP (xylogen and TTS)⇔ Heterogeneous and complex structures complicate their analyses and modifications.
• Various molecular species exist for AGP⇔ Most functions are unknown. Because of the redundancy, changes in phenotypes are unlikely to occur for mutants.

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Arabinogalactan protein as an intercellular signaling molecule

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Visualization of AGP

• The GFP gene is inserted into the core protein gene.
• The expression and localization of specific molecular species can be analyzed.

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Elucidate the physiological functions of AGP sugar chains

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We discovered AGP sugar chain-degrading enzymes and sugar nucleotide synthetase.

*For information of enzymes, visit Expacy’s website (http://enzyme.expasy.org/).

What you can do for yourselves is limited. To advance our research, advice from colleagues and collaborators regarding strategies, useful materials and samples, and interesting findings hidden in failures, are important.
Our research is supported by many people, including Professor Tsumuraya (a distinguished researcher of AGP), my mentor and friends, and collaborators. Seeking their advices when something goes wrong is also a fun in research.