Research activity information

• Mar. 2015 日本農芸化学会, 農芸化学奨励賞, 光合成CO2固定酵素RuBisCOの機能進化研究

  ASHIDA HIROKI

  Japan society


• May 2010 日本生化学会近畿支部, 優秀発表賞, RuBisCO-like proteinを用いた光合成CO2固定酵素RuBisCOの分子進化研究

  ASHIDA HIROKI

  Japan society

• Glycogen deficiency enhances carbon partitioning into glutamate for an alternative extracellular metabolic sink in cyanobacteria.

  Yuichi Kato, Ryota Hidese, Mami Matsuda, Ryudo Ohbayashi, Hiroki Ashida, Akihiko Kondo, Tomohisa Hasunuma

  Glycogen serves as a metabolic sink in cyanobacteria. Glycogen deficiency causes the extracellular release of distinctive metabolites such as pyruvate and 2-oxoglutarate upon nitrogen depletion; however, the mechanism has not been fully elucidated. This study aimed to elucidate the mechanism of carbon partitioning in glycogen-deficient cyanobacteria. Extracellular and intracellular metabolites in a glycogen-deficient ΔglgC mutant of Synechococcus elongatus PCC 7942 were comprehensively analyzed. In the presence of a nitrogen source, the ΔglgC mutant released extracellular glutamate rather than pyruvate and 2-oxoglutarate, whereas its intracellular glutamate level was lower than that in the wild-type strain. The de novo synthesis of glutamate increased in the ΔglgC mutant, suggesting that glycogen deficiency enhanced carbon partitioning into glutamate and extracellular excretion through an unidentified transport system. This study proposes a model in which glutamate serves as the prime extracellular metabolic sink alternative to glycogen when nitrogen is available.

  Feb. 2024, Communications biology, 7(1) (1), 233 - 233, English, International magazine

  Scientific journal


• ppGpp accumulation reduces the expression of the global nitrogen homeostasis-modulating NtcA regulon by affecting 2-oxoglutarate levels

  Ryota Hidese, Ryudo Ohbayashi, Yuichi Kato, Mami Matsuda, Kan Tanaka, Sousuke Imamura, Hiroki Ashida, Akihiko Kondo, Tomohisa Hasunuma

  Abstract The cyanobacterium Synechococcus elongatus PCC 7942 accumulates alarmone guanosine tetraphosphate (ppGpp) under stress conditions, such as darkness. A previous study observed that artificial ppGpp accumulation under photosynthetic conditions led to the downregulation of genes involved in the nitrogen assimilation system, which is activated by the global nitrogen regulator NtcA, suggesting that ppGpp regulates NtcA activity. However, the details of this mechanism have not been elucidated. Here, we investigate the metabolic responses associated with ppGpp accumulation by heterologous expression of the ppGpp synthetase RelQ. The pool size of 2-oxoglutarate (2-OG), which activates NtcA, is significantly decreased upon ppGpp accumulation. De novo ¹³C-labeled CO₂ assimilation into the Calvin-Benson-Bassham cycle and glycolytic intermediates continues irrespective of ppGpp accumulation, whereas the labeling of 2-OG is significantly decreased under ppGpp accumulation. The low 2-OG levels in the RelQ overexpression cells could be because of the inhibition of metabolic enzymes, including aconitase, which are responsible for 2-OG biosynthesis. We propose a metabolic rearrangement by ppGpp accumulation, which negatively regulates 2-OG levels to maintain carbon and nitrogen balance.

  Springer Science and Business Media LLC, Dec. 2023, Communications Biology, 6(1) (1)

  Scientific journal


• Effect of chronic administration with human thioredoxin-1 transplastomic lettuce on diabetic mice.

  Rie Watanabe, Hiroki Ashida, Mikiko Kobayashi-Miura, Akiho Yokota, Junji Yodoi

  Scope: Human thioredoxin-1 (hTrx-1) is a defensive protein induced by various stresses and exerts antioxidative and anti-inflammatory effects. Previously, we described a transplastomic lettuce overexpressing hTrx-1 that exerts a protective effect against oxidative damage in a pancreatic β-cell line. In this study, we treated diabetic mice (Akita mice) with exogenous hTrx-1 and evaluated the effects. Methods and results: Treatment with drinking water and single applications of exogenous hTrx-1 did not influence the feeding, drinking behavior, body weight, blood glucose, or glycosylated hemoglobin (HbA1c) levels in Akita mice. However, chronic administration of a 10% hTrx-1 lettuce-containing diet was associated with a significant reduction from the baseline of HbA1c levels compared with mice fed a wild-type lettuce-containing diet. It also resulted in an increased number of goblet cells in the small intestine, indicating that mucus was synthesized and secreted. Conclusion: Our results revealed that the administration of an hTrx-1 lettuce-containing diet improves the baseline level of HbA1c in Akita mice. This effect is mediated through goblet cell proliferation and possibly related to protection against postprandial hyperglycemia by mucus, which results in the improvement of blood glucose control. These findings suggest that the hTrx-1 lettuce may be a useful tool for the continuous antioxidative and antidiabetic efficacies of the hTrx-1 protein.

  Aug. 2021, Food science & nutrition, 9(8) (8), 4232 - 4242, English, International magazine

  [Refereed]

  Scientific journal


• Reaction of ribulose biphosphate carboxylase/oxygenase assembled on a DNA scaffold

  Huyen Dinh, Eiji Nakata, Peng Lin, Masayuki Saimura, Hiroki Ashida, Takashi Morii

  Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), an enzyme in the Calvin-Benson-Bassham cycle of photosynthesis, catalyzes the first step of CO2 fixation in plants, algae, and photosynthetic bacteria. Despite of the important function in the global carbon cycle, RuBisCO suffers from a slow reaction rate and a competing reaction with O-2 which draw attentions to improve the enzyme efficiency. In this study, a RuBisCO dimer from Rhodospirillum rubrum was assembled on a DNA scaffold using a dimeric DNA binding protein as an adaptor. The enzyme assembly was characterized by atomic force microscopy and RuBisCO assembled on the DNA scaffold showed avid enzymatic activity with retaining its parent carboxylase function. To mimic the environment of the natural microcompartment in cyanobacterial carboxysome that encapsulate the second enzyme carbonic anhydrase (CA) with RuBisCO, RuBisCO was next co-assembled with CA on the DNA scaffold. Although the natural carboxysome assembly is believed to enhance the RuBisCO activity, the co-assembly of RuBisCO and CA reduced the RuBisCO activity, suggesting that the preferential CO2 dehydration by CA reduced the RuBisCO reaction rate. In line with the recent study, our results suggest that the proximity in the interenzyme distance of RuBisCO and CA is not the crucial determinant for the enhanced RuBisCO activity in carboxysome. The assembly of RuBisCO and CA on DNA scaffold provides a platform for further study on the spatial control of RuBisCO and associating enzymes.

  PERGAMON-ELSEVIER SCIENCE LTD, Nov. 2019, BIOORGANIC & MEDICINAL CHEMISTRY, 27(22) (22), 115120 - 115120, English, International magazine

  [Refereed]

  Scientific journal


• Learning RuBisCO's birth and subsequent environmental adaptation

  Hiroki Ashida, Eiichi Mizohata, Akiho Yokota

  It is believed that organisms that first appeared after the formation of the earth lived in a very limited environment, making full use of the limited number of genes. From these early organisms' genes, more were created by replication, mutation, recombination, translocation, and transmission of other organisms' DNA; thus, it became possible for ancient organisms to grow in various environments. The photosynthetic CO2-fixing enzyme RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) began to function in primitive methanogenic archaea and has been evolved as a central CO2-fixing enzyme in response to the large changes in CO2 and O-2 concentrations that occurred in the subsequent 4 billion years. In this review, the processes of its adaptation to be specialized for CO2 fixation will be presented from the viewpoint of functions and structures of RuBisCO.

  PORTLAND PRESS LTD, Feb. 2019, BIOCHEMICAL SOCIETY TRANSACTIONS, 47(1) (1), 179 - 185, English, International magazine

  [Refereed]

  Link to Kobe Univ. Repository (Kernel)


• Increasing photosynthesis/RuBisCO and CO2-concentrating mechanisms

  Hiroki Ashida, Akiho Yokota

  Environmentally friendly energy-producing systems that utilize natural sources of energy, such as solar, wind, land heat, and sea waves, have been developed around the world. These new energy systems are expected to replace fossil fuels for energy production. However, they do not assimilate CO2 liberated into the atmosphere artificially, nor do they produce food or feed for heterotrophs. Thus, it would be prudent to utilize plant systems to synthesize organic matter from inorganic CO2 with solar energy. However, there are intrinsic restrictions on plant production, one of which is the ability of the key photosynthetic enzyme, ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO), to fix CO2 via photosynthesis. Photosynthesis converts CO2 into sugars and lipids. This process relies on biochemical energy in the forms of reduced nicotinamide adenine dinucleotide phosphate and adenosine triphosphate, which are produced from solar energy. As a catalyst, RuBisCO has several disadvantages: it has a low reaction rate, a low affinity for its substrate, CO2, and it catalyzes the oxidation and evolution of carbons in sugars as CO2. There have been many attempts to address these problems, and some have been successful. This chapter provides commentary on recent advances in research and development in this field.

  Elsevier, Jan. 2019, Comprehensive Biotechnology, 169 - 180, English

  In book


• Revisiting the methionine salvage pathway and its paralogues

  Agnieszka Sekowska, Hiroki Ashida, Antoine Danchin

  Methionine is essential for life. Its chemistry makes it fragile in the presence of oxygen. Aerobic living organisms have selected a salvage pathway (the MSP) that uses dioxygen to regenerate methionine, associated to a ratchet-like step that prevents methionine back degradation. Here, we describe the variation on this theme, developed across the tree of life. Oxygen appeared long after life had developed on Earth. The canonical MSP evolved from ancestors that used both predecessors of ribulose bisphosphate carboxylase oxygenase (RuBisCO) and methanethiol in intermediate steps. We document how these likely promiscuous pathways were also used to metabolize the omnipresent by-products of S-adenosylmethionine radical enzymes as well as the aromatic and isoprene skeleton of quinone electron acceptors.

  WILEY, Jan. 2019, MICROBIAL BIOTECHNOLOGY, 12(1) (1), 77 - 97, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea

  Takunari Kono, Sandhya Mehrotra, Chikako Endo, Natsuko Kizu, Mami Matusda, Hiroyuki Kimura, Eiichi Mizohata, Tsuyoshi Inoue, Tomohisa Hasunuma, Akiho Yokota, Hiroyoshi Matsumura, Hiroki Ashida

  Two enzymes are considered to be unique to the photosynthetic Calvin-Benson cycle: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for CO2 fixation, and phosphoribulokinase (PRK). Some archaea possess bona fide RuBisCOs, despite not being photosynthetic organisms, but are thought to lack PRK. Here we demonstrate the existence in methanogenic archaea of a carbon metabolic pathway involving RuBisCO and PRK, which we term 'reductive hexulose-phosphate' (RHP) pathway. These archaea possess both RuBisCO and a catalytically active PRK whose crystal structure resembles that of photosynthetic bacterial PRK. Capillary electrophoresis-mass spectrometric analysis of metabolites reveals that the RHP pathway, which differs from the Calvin-Benson cycle only in a few steps, is active in vivo. Our work highlights evolutionary and functional links between RuBisCO-mediated carbon metabolic pathways in methanogenic archaea and photosynthetic organisms. Whether the RHP pathway allows for autotrophy (that is, growth exclusively with CO2 as carbon source) remains unknown.

  NATURE PUBLISHING GROUP, Jan. 2017, NATURE COMMUNICATIONS, 8, 14007 - 14007, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Potato yield enhancement through intensification of sink and source performances

  Akira Katoh, Hiroki Ashida, Ichiro Kasajima, Shigeru Shigeoka, Akiho Yokota

  The combined total annual yield of six major crops (maize, rice, wheat, cassava, soybean, and potato; Solanum tuberosum L.) amounts to 3.1 billion tons. In recent years, staple crops have begun to be used as substitutes for fossil fuel and feedstocks. The diversion of crop products to fuels and industrial feedstocks has become a concern in many countries because of competition for arable lands and increased food prices. These concerns are definitely justified; however, if plant biotechnology succeeds in increasing crop yields to double the current yields, it will be possible to divert the surplus to purposes other than food without detrimental effects. Maize, rice, wheat, and soybean bear their sink organs in the aerial parts of the plant, and potato in the underground parts. Plants with aerial storage organs cannot accumulate products beyond their capacity to support the weight of these organs. In contrast, potato has heavy storage organs that are supported by the soil. In this mini-review, we introduce strategies of intensifying potato productivity and discuss recent advances in this research area.

  JAPANESE SOC BREEDING, Jan. 2015, BREEDING SCIENCE, 65(1) (1), 77 - 84, English, Domestic magazine

  [Refereed]

  Scientific journal


• Plausible Novel Ribose Metabolism Catalyzed by Enzymes of the Methionine Salvage Pathway in Bacillus subtilis

  Toshihiro Nakano, Yohtaro Saito, Akiho Yokota, Hiroki Ashida

  The methionine salvage pathway (MSP) recycles reduced sulfur from 5-methylthioribose. Here we propose a novel ribose metabolic pathway performed by MSP enzymes of Bacilli. MtnK, an initial catalyst of MSP, had significant ribose kinase activity, with V-max and K-m values of 2.9 mu mol min(-1) mg of protein(-1) and 4.8 mm. Downstream enzymes catalyzed the isomerization of ribose-1-phosphate and subsequent dehydration, enolization, dephosphorylation, and dioxygenation.

  TAYLOR & FRANCIS LTD, May 2014, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77(5) (5), 1104 - 1107, English, International magazine

  [Refereed]

  Scientific journal


• Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme

  Wonchull Kang, Se Hoon Hong, Hye Min Lee, Na Yeon Kim, Yun Chan Lim, Le Thi My Le, Bitna Lim, Hyun Chul Kim, Tae Yeon Kim, Hiroki Ashida, Akiho Yokota, Sang Soo Hah, Keun Ho Chun, Yong-Keun Jung, Jin Kuk Yang

  APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a K-m of 9.32 mu M and a V-max of 1.39 mu mol min(-1) mg(-1). The crystal structure was determined at 2.0-angstrom resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.

  NATL ACAD SCIENCES, Jan. 2014, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 111(1) (1), E54 - E61, English, International magazine

  [Refereed]

  Scientific journal


• MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of Tetrahymena thermophila

  Toshihiro Nakano, Izuru Ohki, Akiho Yokota, Hiroki Ashida

  To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and H-1-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.

  PUBLIC LIBRARY SCIENCE, Dec. 2013, PLOS ONE, 8(7) (7), e67385, English, International magazine

  [Refereed]

  Scientific journal


• His267 is involved in carbamylation and catalysis of RuBisCO-like protein from Bacillus subtilis

  Toshihiro Nakano, Yohtaro Saito, Akiho Yokota, Hiroki Ashida

  Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) from Bacillus subtilis catalyze mechanistically similar enolase reactions. Both enzymes require carbamylation of the e-amino group of the active site lysine during activation to generate the binding site of the essential Mg2+ ion. His267 forms a possible hydrogen bond with the carbamate of the active site Lys176 in B. subtilis RLP. This active site histidine is completely conserved in RLPs and RuBisCO. H267Q, H267N and H267A mutant enzymes required higher CO2 concentrations for maximal activity than wild-type enzyme, suggesting that the histidine is involved in high affinity for activator CO2 in Bacillus RLP. These mutations showed weak effects on the catalysis of RLP, whereas this residue is reportedly essential for catalysis in RuBisCO but is not involved in the carbamylation. The different functions of the active site histidine in RLP and RuBisCO are discussed. (C) 2013 Elsevier Inc. All rights reserved.

  ACADEMIC PRESS INC ELSEVIER SCIENCE, Feb. 2013, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 431(2) (2), 176 - 180, English, International magazine

  [Refereed]

  Scientific journal


• Traveling waves of circadian gene expression in lettuce

  Kazuya Ukai, Koji Inai, Norihito Nakamichi, Hiroki Ashida, Akiho Yokota, Yusuf Hendrawan, Haruhiko Murase, Hirokazu Fukuda

  The circadian clock controls many physiological states in plants by synchronization with environmental changes. The clock can be controlled artificially by imposing various light conditions in a closed system for plant cultivation. The plant circadian rhythm is formed by enormous self-sustained cellular circadian oscillators, so that the synchronization of circadian oscillators is of great importance to the formation of individual circadian rhythms. Therefore, the study of synchronization phenomena is important for precise control of the circadian rhythm. In this study, we have investigated the spatiotemporal dynamics of circadian oscillators in lettuce (Lactuca sativa L.). Bioluminescence of transgenic lettuce carrying a CCA1::LUC construct as a reporter of a circadian gene expression was measured in the leaf and root. We observed phase wave propagation in the leaf with a phase delay in the region of the primary vein, with the primary vein region showing lower amplitude than the other regions. Wave propagation occurred from the edge of the leaf inward. In addition, a striped wave traveled from the base to the tip along the roots, which were grown under continuous dark or light conditions. The features observed in the lettuce plants could be explained by phase oscillator models established in the study of Arabidopsis thaliana. The results of this study show the possibility of applying circadian clock control in a model plant for plant production.

  2012, Environmental Control in Biology, 50(3) (3), 237 - 246, English

  [Refereed]

  Scientific journal


• Increasing Photosynthesis/RuBisCO and CO2-Concentrating Mechanisms

  H. Ashida, A. Yokota

  Environmentally friendly energy-producing systems that utilize natural sources of energy, such as solar, wind, land heat, and sea waves, have been developed around the world. These new energy systems are expected to replace fossil fuels for energy production. However, they do not assimilate CO2 liberated into the atmosphere artificially, nor do they produce food or feed for heterotrophs. Thus, it would be prudent to utilize plant systems to synthesize organic matter from inorganic CO2 with solar energy. However, there are intrinsic restrictions on plant production, one of which is the ability of the key photosynthetic enzyme, ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO), to fix CO2 via photosynthesis. Photosynthesis converts CO2 into sugars and lipids. This process relies on biochemical energy in the forms of reduced nicotinamide adenine dinucleotide phosphate and adenosine triphosphate, which are produced from solar energy. As a catalyst, RuBisCO has several disadvantages: it has a low reaction rate, a low affinity for its substrate, CO2, and it catalyzes the oxidation and evolution of carbons in sugars as CO2. There have been many attempts to address these problems, and some have been successful. This article provides commentary on recent advances in research and development in this field.

  Elsevier Inc., Sep. 2011, Comprehensive Biotechnology, Second Edition, 4, 165 - 176, English

  [Refereed]

  In book


• Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

  Soon Lim, Hiroki Ashida, Rie Watanabe, Koji Inai, Yun-Soo Kim, Keiko Mukougawa, Hirokazu Fukuda, Ken-ichi Tomizawa, Kei-ichi Ushiyama, Hiroshi Asao, Masahiro Tamoi, Hiroshi Masutani, Shigeru Shigeoka, Junji Yodoi, Akiho Yokota

  The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.

  SPRINGER, Jul. 2011, PLANT MOLECULAR BIOLOGY, 76(3-5) (3-5), 335 - 344, English, International magazine

  [Refereed]

  Scientific journal


• Generation of transplastomic lettuce with enhanced growth and high yield.

  Yaka Ichikawa, Masahiro Tamoi, Harumi Sakuyama, Takanori Maruta, ASHIDA HIROKI, Akiho Yokota, Shigeru Shigeoka

  Nov. 2010, GM Crops, 1, 322 - 326, English

  [Refereed]

  Scientific journal


• A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA

  Kenji Nishimura, Hiroki Ashida, Taro Ogawa, Akiho Yokota

  P>In plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes post-maturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.

  WILEY, Sep. 2010, PLANT JOURNAL, 63(5) (5), 766 - 777, English, International magazine

  [Refereed]

  Scientific journal


• An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein

  Toshihiro Nakano, Hiroki Ashida, Eiichi Mizohata, Hiroyoshi Matsumura, Akiho Yokota

  Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the beta-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k(eat) for carboxylase activity, a 2-fold increase in K-m for CO2, and a 1.9-fold increase in K-m for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k(cat) and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct. (C) 2010 Elsevier Inc. All rights reserved.

  ACADEMIC PRESS INC ELSEVIER SCIENCE, Feb. 2010, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 392(2) (2), 212 - 216, English, International magazine

  [Refereed]

  Scientific journal


• Structure of the apo decarbamylated form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis

  Haruka Tamura, Yohtaro Saito, Hiroki Ashida, Yasushi Kai, Tsuyoshi Inoue, Akiho Yokota, Hiroyoshi Matsumura

  2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase), a RuBisCO-like protein (RLP), catalyzes the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate. The crystal structure of the apo decarbamylated form (E form) of Bacillus subtilis DK-MTP-1P enolase (Bs-DK-MTP-1P enolase) has been determined at 2.3 A resolution. The overall structure of the E form of Bs-DK-MTP-1P enolase highly resembles that of Geobacillus kaustophilus DK-MTP-1P enolase (Gk-DK-MTP-1P enolase), with the exception of a few insertions or deletions and of a few residues at the active site. In the E form of Bs-DK-MTP-1P enolase, Lys150 (equivalent to Lys175 in RuBisCO) at the active site adopts a conformation that is distinct from those observed in the other forms of Gk-DK-MTP-1P enolase. This unusual conformational change appears to be induced by changes in the phi and epsilon angles of Gly151, which is conserved in the sequences of the Bs-DK-MTP-1P and Gk-DK-MTP-1P enolases but not in those of RuBisCOs. The loop at 303-312, equivalent to the catalytic loop termed `loop-6' in RuBisCO, is in a closed conformation in the E form of Bs-DK-MTP-1P enolase. The closed conformation appears to be stabilized by Pro312, which is conserved in the sequences of several RLPs (equivalent to Glu338 in RuBisCO). Based on these results, the characteristic structural features of DK-MTP-1P enolase are discussed.

  INT UNION CRYSTALLOGRAPHY, Sep. 2009, ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 65, 942 - 951, English

  [Refereed]

  Scientific journal


• A Phosphofructokinase B-Type Carbohydrate Kinase Family Protein, NARA5, for Massive Expressions of Plastid-Encoded Photosynthetic Genes in Arabidopsis

  Taro Ogawa, Kenji Nishimura, Takehiko Aoki, Hisabumi Takase, Ken-Ichi Tomizawa, Hiroki Ashida, Akiho Yokota

  To date, there have been no reports on screening for mutants defective in the massive accumulation of Rubisco in higher plants. Here, we describe a screening method based on the toxic accumulation of ammonia in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase, during photorespiration initiated by the oxygenase reaction of Rubisco in Arabidopsis (Arabidopsis thaliana). Five recessive mutants with decreased amounts of Rubisco were identified and designated as nara mutants, as they contained a mutation in genes necessary for the achievement of Rubisco accumulation. The nara5-1 mutant showed markedly lower levels of plastid-encoded photosynthetic proteins, including Rubisco. Map-based cloning revealed that NARA5 encoded a chloroplast phosphofructokinase B-type carbohydrate kinase family protein of unknown function. The NARA5 protein fused to green fluorescent protein localized in chloroplasts. We conducted expression analyses of photosynthetic genes during light-induced greening of etiolated seedlings of nara5-1 and the T-DNA insertion mutant, nara5-2. Our results strongly suggest that NARA5 is indispensable for hyperexpression of photosynthetic genes encoded in the plastid genome, particularly rbcL.

  AMER SOC PLANT BIOLOGISTS, Sep. 2009, PLANT PHYSIOLOGY, 151(1) (1), 114 - 128, English, International magazine

  [Refereed]

  Scientific journal


• Structure of the apo decarbamylated form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis.

  Haruka Tamura, Yohtaro Saito, Hiroki Ashida, Yasushi Kai, Tsuyoshi Inoue, Akiho Yokota, Hiroyoshi Matsumura

  2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase), a RuBisCO-like protein (RLP), catalyzes the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate. The crystal structure of the apo decarbamylated form (E form) of Bacillus subtilis DK-MTP-1P enolase (Bs-DK-MTP-1P enolase) has been determined at 2.3 A resolution. The overall structure of the E form of Bs-DK-MTP-1P enolase highly resembles that of Geobacillus kaustophilus DK-MTP-1P enolase (Gk-DK-MTP-1P enolase), with the exception of a few insertions or deletions and of a few residues at the active site. In the E form of Bs-DK-MTP-1P enolase, Lys150 (equivalent to Lys175 in RuBisCO) at the active site adopts a conformation that is distinct from those observed in the other forms of Gk-DK-MTP-1P enolase. This unusual conformational change appears to be induced by changes in the varphi and psi angles of Gly151, which is conserved in the sequences of the Bs-DK-MTP-1P and Gk-DK-MTP-1P enolases but not in those of RuBisCOs. The loop at 303-312, equivalent to the catalytic loop termed ;loop-6' in RuBisCO, is in a closed conformation in the E form of Bs-DK-MTP-1P enolase. The closed conformation appears to be stabilized by Pro312, which is conserved in the sequences of several RLPs (equivalent to Glu338 in RuBisCO). Based on these results, the characteristic structural features of DK-MTP-1P enolase are discussed.

  Sep. 2009, Acta crystallographica. Section D, Biological crystallography, 65(Pt 9) (Pt 9), 942 - 51, English, International magazine

  [Refereed]

  Scientific journal


• Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

  Yohtaro Saito, Hiroki Ashida, Tomoko Sakiyama, Nicole Tandeau de Marsac, Antoine Danchin, Agnieszka Sekowska, Akiho Yokota

  The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO2 fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg2+ for catalysis and, like RuBisCO, was stimulated by CO2. Four residues that are essential for the enolization reaction of RuBisCO, Lys175, Lys201, Asp203, and Glu204, were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys123, the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K-i value of 103 mu M. RuBP and D-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.

  AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, May 2009, JOURNAL OF BIOLOGICAL CHEMISTRY, 284(19) (19), 13256 - 13264, English, International magazine

  [Refereed]

  Scientific journal


• Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis

  Haruka Tamura, Hiroki Ashida, Shogo Koga, Yohtaro Saito, Tomonori Yadani, Yasushi Kai, Tsuyoshi Inoue, Akiho Yokota, Hiroyoshi Matsumura

  2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 angstrom resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P2(1), with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 angstrom, beta = 90.8 degrees. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V-M value of 2.2 angstrom(3) Da(-1) and a solvent content of 43%.

  INT UNION CRYSTALLOGRAPHY, Feb. 2009, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, 65(Pt 2) (Pt 2), 147 - 150, English, International magazine

  [Refereed]

  Scientific journal


• RuBisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RuBisCO-like proteins and photosynthetic RuBisCO

  Hiroki Ashida, Yohtaro Saito, Toshihiro Nakano, Nicole Tandeau de Marsac, Agnieszka Sekowska, Antoine Danchin, Akiho Yokota

  Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme in the fixation of CO2 in the Calvin cycle of plants. Many genome projects have revealed that bacteria, including Bacillus subtilis, possess genes for proteins that are similar to the large subunit of RuBisCO. These RuBisCO homologues are called RuBisCO-like proteins (RLPs) because they are not able to catalyse the carboxylase or the oxygenase reactions that are catalysed by photosynthetic RuBisCO. It has been demonstrated that B. subtilis RLP catalyses the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. The structure of DK-MTP-1-P is very similar to that of ribulose-1,5-bisphosphate (RuBP) and the enolase reaction is a part of the reaction catalysed by photosynthetic RuBisCO. In this review, functional and evolutionary relationships between B. subtilis RLP of the methionine salvage pathway, other RLPs, and photosynthetic RuBisCO are discussed. In addition, the fundamental question, 'How has RuBisCO evolved?' is also considered, and evidence is presented that RuBisCOs evolved from RLPs.

  OXFORD UNIV PRESS, May 2008, JOURNAL OF EXPERIMENTAL BOTANY, 59(7) (7), 1543 - 1554, English, International magazine

  [Refereed]

  Scientific journal


• Enzymatic characterization of 5-methylthioribulose-1-phosphate dehydratase of the methionine salvage pathway in Bacillus subtilis

  Hiroki Ashida, Yohtaro Saito, Chojiro Kojinia, Akiho Yokota

  5-Methylthioribulose-1-phosphate (MTRu-1-P) dehydratase catalyzes the reaction from MTRu-1-P to 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) in the methionine salvage pathway in Bacillus subtilis. The properties of this enzyme remain to be determined. We characterized these properties using a recombinant protein. The enzyme, with a molecular mass of 90 kDa, was composed of four subunits. The K-m and V-max of the enzyme were 8.9 mu M and 42.7 mu mole min(-1) mg protein(-1) at 25 degrees C respectively. Maximum activity was observed at pH 7.5 to 8.5 and 40 degrees C. The activation energy of the reaction from MTRu-1-P to DK-MTP-1-P was 63.5 kJ mol-1. The reaction product DK-MTP-1-P was labile, and decomposed at a rate constant of 0.048 s(-1) to an unknown compound that was not utilized by DK-MTP-1-P enolase, the enzyme catalyzing the next step. The function of this enzyme in the pathway is discussed.

  TAYLOR & FRANCIS LTD, Apr. 2008, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72(4) (4), 959 - 967, English

  [Refereed]

  Scientific journal


• Crystal structure of RuBisCO-like protein from Bacillus subtilis

  Haruka Tamura, Yohtaro Saito, Hiroki Ashida, Hiroyoshi Matsumura, Yasushi Kai, Akiho Yokota, Tsuyoshi Inoue

  INT UNION CRYSTALLOGRAPHY, 2008, ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES, 64, C261 - C262, English

  [Refereed]


• Crystal structure of 5-methylthioribose 1-phosphate isomerase product complex from Bacillus subtilis: Implications for catalytic mechanism

  Haruka Tamura, Yohtaro Saito, Hiroki Ashida, Tsuyoshi Inoue, Yasushi Kai, Akiho Yokota, Hiroyoshi Matsumura

  The methionine salvage pathway (MSP) plays a crucial role in recycling a sulphahydryl derivative of the nucleoside. Recently, the genes and reactions in MSP from Bacillus subtilis have been identified, where 5-methylthioribose 1-phosphate isomerase (M1Pi) catalyzes a conversion of 5-methylthioribose 1- phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). Herein, we report the crystal structures of B. subtilis M1Pi (Bs-M1Pi) in complex with its product MTRu-1-P, and a sulfate at 2.4 and 2.7 angstrom resolution, respectively. The electron density clearly shows the presence of each compound in the active site. The structural comparison with other homologous proteins explains how the substrate uptake of Bs- M1Pi may be induced by an open/closed transition of the active site. The highly conserved residues at the active site, namely, Cys160 and Asp240 are most likely to be involved in catalysis. The structural analysis sheds light on its catalytic mechanism of M1Pi.

  WILEY-BLACKWELL, Jan. 2008, PROTEIN SCIENCE, 17(1) (1), 126 - 135, English

  [Refereed]

  Scientific journal


• Molecular mechanisms of RuBisCO biosynthesis in higher plants

  Kenji Nishimura, Taro Ogawa, Hiroki Ashida, Akiho Yokota

  Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyses the initial step of photosynthetic CO 2 assimilation, although its catalytic efficiency is very low. Therefore, higher plants must synthesize large amounts of RuBisCO to compensate for its inefficient enzymatic properties. The holoenzyme of RuBisCO consists of 8 large and 8 small subunits, whose genes are individually encoded on two distinct genomes located in the chloroplast and the nucleus, respectively. RuBisCO biosynthesis requires many factors involved in transcription, translation, folding and assembly processes. However, the mechanisms underlying these processes are complex, and therefore the molecular mechanisms and regulation systems of RuBisCO biosynthesis are not yet fully understood. In this review, we introduce recent research on the molecular mechanisms of RuBisCO biosynthesis in higher plants, and discuss future perspectives in this field of research.

  JAPANESE SOC PLANT CELL & MOLECULAR BIOL, 2008, PLANT BIOTECHNOLOGY, 25(3) (3), 285 - 290, English

  [Refereed]

  Scientific journal


• Enzymatic characterization of 5-methylthioribose 1-Phosphate isomerase from bacillus subtilis

  Yohtaro Saito, Hiroki Ashida, Chopro Kojlma, Haruka Tamura, Hiroyoshi Matsumura, Yasushi Kai, Akiho Yokota

  The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P? of 138 mu M, and showed that the maximum velocity of the reaction was 20.4 mu mol min(-1) (mg protein)(-1). The optimum reaction temperature and reaction pH were 35 degrees C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol(-1). The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.

  TAYLOR & FRANCIS LTD, Aug. 2007, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71(8) (8), 2021 - 2028, English

  [Refereed]

  Scientific journal


• Improvement of cyanobacterial RuBisCO by introducing the latch structure of red algal RuBisCO with high specificity for CO2 fixation.

  Nana Ninomiya, Hiroki Ashida, Akiho Yokota

  OXFORD UNIV PRESS, 2007, PLANT AND CELL PHYSIOLOGY, 48, S71 - S71, English

  [Refereed]


• Characterization of Arabidopsis mutants for quantitative regulation of RuBisCO.

  Taro Ogawa, Takehiko Aoki, Hiroki Ashida, Akiho Yokota

  OXFORD UNIV PRESS, 2007, PLANT AND CELL PHYSIOLOGY, 48, S214 - S214, English

  [Refereed]


• A new Rubisco-like protein coexists with a photosynthetic Rubisco in the planktonic cyanobacteria Microcystis

  Alyssa Carre-Mlouka, Annick Mejean, Philippe Quillardet, Hiroki Ashida, Yohtaro Saito, Akiho Yokota, Isabelle Callebaut, Agnieszka Sekowska, Elke Dittmann, Christiane Bouchier, Nicole Tandeau de Marsac

  Two genes encoding proteins related to large subunits of Rubisco were identified in the genome of the planktonic cyanobacterium Microcystis aeruginosa PCC 7806 that forms water blooms worldwide. The rbcL(I) gene belongs to the form I subfamily typically encountered in cyanobacteria, green algae, and land plants. The second and newly discovered gene is of the form IV subfamily and widespread in the Microcystis genus. In M. aeruginosa PCC 7806 cells, the expression of both rbcL(I) and rbcL(IV) is sulfur-dependent. The purified recombinant RbcL(IV) overexpressed in Escherichia coli cells did not display CO2 fixation activity but catalyzed enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate, and the rbcL(IV) gene rescued a Bacillus subtilis MtnW-deficient mutant. Therefore, the Microcystis RbcL(IV) protein functions both in vitro and in vivo and might be involved in a methionine salvage pathway. Despite variations in the amino acid sequences, RbcL(IV) shares structural similarities with all members of the Rubisco superfamily. Invariant amino acids within the catalytic site may thus represent the minimal set for enolization, whereas variations, especially located in loop 6, may account for the limitation of the catalytic reaction to enolization. Even at low protein concentrations in vitro, the recombinant RbcL(IV) assembles spontaneously into dimers, the minimal unit required for Rubisco forms I-III activity. The discovery of the coexistence of RbcL(I) and RbcL(IV) in cyanobacteria, the ancestors of chloroplasts, enlightens episodes of the chaotic evolutionary history of the Rubiscos, a protein family of major importance for life on Earth.

  AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Aug. 2006, JOURNAL OF BIOLOGICAL CHEMISTRY, 281(34) (34), 24462 - 24471, English

  [Refereed]

  Scientific journal


• Screening of RuBisCO biosynthesis mutants in Arabidopsis thaliana.

  Ogawa Taro, Asida Hiroki, Takase Naobumi, Tomizawa Ken-ichi, Yokota Akiho

  Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme in photosynthetic CO₂ fixation. In higher plants, RuBisCO is a multimeric enzyme composed of large and small subunits encoded by the chloroplast rbcL gene and nuclear rbcS genes. RuBisCO is biosynthesized via complex processes, hence overall mechanism is still unknown. To understand the molecular mechanism for these processes, we have designed a positive method for screening RuBisCO biosynthesis mutants using methionine sulfoximine (MSX) which is an inhibitor of glutamine synthetase. Wild-type plants treated with MSX could not survive because photorespiratory NH₃ accumulated massively depending on RuBisCO oxygenase activity. In contrast, rca mutants which lacked the activation of RuBisCO could survive under the same condition. After screened of 11000 EMS mutagenized seedlings, we obtained eight mutants which show either decreased RuBisCO amounts or low activities. These results indicate that this system is useful for screening of RuBisCO biosynthesis mutants.

  The Japanese Society of Plant Physiologists, 2006, Plant and Cell Physiology Supplement, 2006, 119 - 119


• Was photosynthetic RuBisCO recruited by acquisitive evolution from RuBisCO-like proteins involved in sulfur metabolism?

  H Ashida, A Danchin, A Yokota

  Genome analyses have revealed that the genomes of non-photosynthetic bacteria including Bacillus subtilis code for proteins similar to the large subunit of RuBisCO (called RuBisCO-like protein (RLP)). This raises a fundamental question as to their functional relationship to photosynthetic RuBisCO. Recently, we identified the RLP of B. subtilis as the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase in the methionine salvage pathway. In this mini-review, we suggest functional and evolutionary links between B. subtilis RLP and photosynthetic RuBisCO. Furthermore, we propose that photosynthetic RuBisCOs evolved from RLPs similar to that found in B. subtilis. (c) 2005 Elsevier SAS. All rights reserved.

  ELSEVIER SCIENCE BV, Jun. 2005, RESEARCH IN MICROBIOLOGY, 156(5-6) (5-6), 611 - 618, English

  [Refereed]

  Scientific journal


• Crystallization and preliminary X-ray analysis of methylthioribose-1-phosphate isomerase from Bacillus subtilis

  H Tamura, H Matsumura, T Inoue, H Ashida, Y Saito, A Yokota, Y Kai

  BLACKWELL PUBLISHING, Jun. 2005, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 61, 595 - 598, English

  [Refereed]

  Scientific journal


• Crystal Structure of Methylthioribose-1-phosphate Isomerase from Bacillus subtilis

  Haruka Tamura, Hiroyoshi Matsumura, Tsuyoshi Inoue, Hiroki Ashida, Yohtaro Saito, Akiho Yokota, Yasushi Kai

  INT UNION CRYSTALLOGRAPHY, 2005, ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES, 61, C207 - C208, English

  [Refereed]


• Bacterial variations on the methionine salvage pathway

  A Sekowska, Denervaud, V, H Ashida, K Michoud, D Haas, A Yokota, A Danchin

  Background: The thiomethyl group of S-adenosylmethionine is often recycled as methionine from methylthioadenosine. The corresponding pathway has been unravelled in Bacillus subtilis. However methylthioadenosine is subjected to alternative degradative pathways depending on the organism. Results: This work uses genome in silico analysis to propose methionine salvage pathways for Klebsiella pneumoniae, Leptospira interrogans, Thermoanaerobacter tengcongensis and Xylella fastidiosa. Experiments performed with mutants of B. subtilis and Pseudomonas aeruginosa substantiate the hypotheses proposed. The enzymes that catalyze the reactions are recruited from a variety of origins. The first, ubiquitous, enzyme of the pathway, MtnA (methylthioribose-1-phosphate isomerase), belongs to a family of proteins related to eukaryotic intiation factor 2B alpha. mtnB codes for a methylthioribulose-1-phosphate dehydratase. Two reactions follow, that of an enolase and that of a phosphatase. While in B. subtilis this is performed by two distinct polypeptides, in the other organisms analyzed here an enolase-phosphatase yields 1,2-dihydroxy-3-keto-5-methylthiopentene. In the presence of dioxygen an aci-reductone dioxygenase yields the immediate precursor of methionine, ketomethylthiobutyrate. Under some conditions this enzyme produces carbon monoxide in B. subtilis, suggesting a route for a new gaseous mediator in bacteria. Ketomethylthiobutyrate is finally transaminated by an aminotransferase that exists usually as a broad specificity enzyme (often able to transaminate aromatic aminoacid keto-acid precursors or histidinol-phosphate). Conclusion: A functional methionine salvage pathway was experimentally demonstrated, for the first time, in P. aeruginosa. Apparently, methionine salvage pathways are frequent in Bacteria ( and in Eukarya), with recruitment of different polypeptides to perform the needed reactions (an ancestor of a translation initiation factor and RuBisCO, as an enolase, in some Firmicutes). Many are highly dependent on the presence of oxygen, suggesting that the ecological niche may play an important role for the existence and/or metabolic steps of the pathway, even in phylogenetically related bacteria. Further work is needed to uncover the corresponding steps when dioxygen is scarce or absent (this is important to explore the presence of the pathway in Archaea). The thermophile T. tengcongensis, that thrives in the absence of oxygen, appears to possess the pathway. It will be an interesting link to uncover the missing reactions in anaerobic environments.

  BIOMED CENTRAL LTD, Mar. 2004, BMC MICROBIOLOGY, 4, English

  [Refereed]

  Scientific journal


• A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO

  H Ashida, Y Saito, C Kojima, K Kobayashi, N Ogasawara, A Yokota

  The genomes of several nonphotosynthetic bacteria, such as Bacillus subtilis, and some Archaea include genes for proteins with sequence homology to the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO). We found that such a RuBisCO-like protein (RLP) from B. subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway. A growth-defective mutant, in which the gene for this RLP had been disrupted, was rescued by the gene for RuBisCO from the photosynthetic bacterium Rhodospirillum rubrum. Thus, the photosynthetic RuBisCO from R. rubrum retains the ability to function in the methionine salvage pathway in B. subtilis.

  AMER ASSOC ADVANCEMENT SCIENCE, Oct. 2003, SCIENCE, 302(5643) (5643), 286 - 290, English

  [Refereed]

  Scientific journal


• Crystal structure of activated ribulose-1,5-bisphosphate carboxylase/oxygenase from green alga Chlamydomonas reinhardtii complexed with 2-carboxyarabinitol-1,5-bisphosphate

  E Mizohata, H Matsumura, Y Okano, M Kumei, H Takuma, J Onodera, K Kato, N Shibata, T Inoue, A Yokota, Y Kai

  Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO2 and O-2, respectively, with ribulose-1,5-bisphosphate. Many photosynthetic organisms have form 1 rubiscos comprised of eight large (L) and eight small (S) subunits. The crystal structure of the complex of activated rubisco from the green alga Chlamydomonas reinliardtii and the reaction intermediate analogue 2-carboxyarabinitol-1,5-bisphosphate (2-CABP) has been solved at 1.84 Angstrom resolution (R-cryst of 15.2% and R-free of 18.1%). The subunit arrangement of Chlamydomonas rubisco is the same as those of the previously solved form I rubiscos. Especially, the present structure is very similar to the activated spinach structure complexed with 2-CABP in the L-subunit folding and active-site conformation, but differs in S-subunit folding. The central insertion of the Chlamydomonas S-subunit forms the longer betaA-betaB loop that protrudes deeper into the solvent channel of rubisco than higher plant, cyanobacterial, and red algal (red-like) betaA-betaB loops. The C-terminal extension of the Chlamydomonas S-subunit does not protrude into the solvent channel, unlike that of the red algal S-subunit, but lies on the protein surface anchored by interactions with the N-terminal region of the S-subunit. Further, the present high-resolution structure has revealed novel post-translational modifications. Residue 1 of the S-subunit is N-alpha-methylmethionine, residues 104 and 151 of the L-subunit are 4-hydroxyproline, and residues 256 and 369 of the L-subunit are S-gamma-methylcysteine. Furthermore, the unusual electron density of residue 471 of the L-subunit, which has been deduced to be threonine from the genomic DNA sequence, suggests that the residue is isoleucine produced by RNA editing or O-gamma-methylthreonine. (C) 2002 Elsevier Science Ltd.

  ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, Feb. 2002, JOURNAL OF MOLECULAR BIOLOGY, 316(3) (3), 679 - 691, English

  [Refereed]

  Scientific journal

• 低分子抗酸化物含有食による糖尿病モデルマウス血糖コントロールの改善

  渡邉理江, 蘆田弘樹, 増谷弘, 三浦美樹子, 三浦美樹子, 横田明穂, 淀井淳司, 淀井淳司

  (株)インフォノーツパブリッシング, Dec. 2019, 機能性食品と薬理栄養, 13(3) (3), 180 - 180, Japanese

  Summary national conference


• 二酸化炭素の環境問題と利用 光合成二酸化炭素固定酵素RuBisCO

  蘆田弘樹

  01 Nov. 2018, Journal of the Society of Inorganic Materials, Japan, 25(397) (397), 406‐411, Japanese


• メタン生成アーキアにおけるRuBisCOを利用した新規CO₂固定経路

  蘆田弘樹

  Oct. 2017, 酵素工学ニュ-ス, (78) (78), 14‐18, Japanese


• 多彩な戦略で挑むシアノバクテリア由来の燃料生産 持続可能な第三世代バイオ燃料生産の最前線

  日原由香子, 朝山宗彦, 蘆田弘樹, 天尾豊, 新井宗仁, 粟井光一郎, 得平茂樹, 小山内崇, 鞆達也, 成川礼, 蓮沼誠久, 増川一

  日本農芸化学会 ; 1962-, 20 Jan. 2017, 化学と生物, 55(2) (2), 88‐97 - 97, Japanese


• CO₂資源化:研究者たちの挑戦 4 CO₂資源化を目指した光合成炭素固定酵素RuBisCOの機能進化研究

  蘆田弘樹

  日本化学会, 01 Nov. 2016, 化学と工業, 69(11) (11), 957‐959 - 959, Japanese


• ヒトチオレドキシン-1高発現レタス長期食餌負荷による糖尿病モデルマウス血糖コントロールの改善

  渡邉 理江, 蘆田 弘樹, 増谷 弘, 三浦 美樹子[小林], 横田 明穂, 淀井 淳司

  (公社)日本生化学会, Dec. 2015, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [1P0546] - [1P0546], English


• ジャガイモ塊茎を形成するストロンの原基である地中腋芽におけるRanGTPase1遺伝子の発現解析

  KATO AKIRA, FUKUI YUKI, ASHIDA HIROKI, AKASHI KIN'YA, KAJIKAWA MASATAKA, SHIGEOKA SHIGERU, YOKOTA AKIHO

  05 Mar. 2014, 日本農芸化学会大会講演要旨集(Web), 2014, 2C04A08 (WEB ONLY), Japanese


• シアノバクテリアの光合成能力を利用したバイオ燃料生産

  蘆田弘樹

  日本生物工学会, 25 Jun. 2013, 生物工学会誌, 91(6) (6), 352 - 352, Japanese


• シアノバクテリアの光合成能力を利用したバイオ燃料生産

  ASHIDA HIROKI

  Jun. 2013, 生物工学会誌, 91(6) (6), 352, Japanese

  [Invited]

  Introduction scientific journal


• レタス葉緑体における医療用タンパク質ヒトチオレドキシン1の生産

  ASHIDA HIROKI, LIM SOON, WATANABE RIE, INAI KOJI, KIM YUN-SOO, MUKAIGAWA KEIKO, FUKUDA HIROKAZU, TAMOI MASAHIRO, MASUTANI HIROSHI, SHIGEOKA SHIGERU, YODOI JUNJI, YOKOTA AKIHO

  The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Human thioredoxin 1 (hTrx1) is an antioxidant protein, and can suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a human therapeutic protein.
  We created transplastomic lettuce expressing hTrx1 gene under the control of the tobacco psbA promoter. The hTrx1 protein accumulated to approximately 3% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected MIN6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 form Escherichia coli. This is the first report for expression of the biologically active hTrx1 protein in plant chloroplasts. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.

  The Japanese Society of Plant Physiologists, 11 Mar. 2011, 日本植物生理学会年会要旨集, 52nd, 129 - 75, Japanese


• 光合成CO₂固定酵素RuBisCO誕生の秘密 RuBisCO‐like proteinが隠しもつRuBisCOとしての潜在能力

  齋藤洋太郎, 蘆田弘樹

  Japan Society for Bioscience, Biotechnology, and Agrochemistry, 01 Nov. 2010, 化学と生物, 48(11) (11), 739 - 742, Japanese


• 医・農・工融合によるヒトチオレドキシン1産生レタスの生産技術の開発

  ASHIDA HIROKI, TAMOI MASAHIRO, FUKUDA HIROKAZU, WATANABE RIE, SOON LIM, INAI KOJI, MUKAIGAWA KEIKO, YAMAKAWA HIRONOBU, KATO MASAO, USHIYAMA KEIICHI, MASUTANI HIROSHI, SHIGEOKA SHIGERU, YODOI JUNJI, YOKOTA AKIHO

  29 Sep. 2010, バイオテクノロジーシンポジウム予稿集, 28th, 56 - 57, Japanese


• 光合成CO2固定酵素RuBisCOとRuBisCO-like Proteinの比較研究が明らかにしたこと--RuBisCOはどのようにCO2固定能を獲得したのか? (酵素工学研究会第59回講演会)

  蘆田 弘樹, 横田 明穗

  酵素工学研究会, 25 Apr. 2008, 酵素工学研究会講演会講演要旨集, 59, 11 - 18, Japanese


• Rubisco satellite meeting Reaserch Frontiers with Rubisco, the “Elixir of Life” in the Biospherに参加して

  蘆田 弘樹

  Dec. 2007, 光合成研究, 17, 84 - 86

  [Invited]


• 医・農・工融合によるヒトチオレドキシン1産生レタスの生産技術の開発

  YOKOTA AKIHO, SHIGEOKA SHIGERU, YODOI JUNJI, ASHIDA HIROKI, TAMOI MASAHIRO, HISAZUMI TAKAAKI, FUKUDA HIROKAZU, KATO NORIKO, SOON LIM, INAI KOJI, MASUTANI HIROSHI, AKASHI KIN'YA

  06 Nov. 2007, バイオテクノロジーシンポジウム予稿集, 25th, 117 - 118, Japanese


• Improvement of cyanobacterial RuBisCO by introducing the latch structure involved in high affinity for CO2 in red algal RuBisCO.

  N. Ninomiya, H. Ashida, A. Yokota

  SPRINGER, Feb. 2007, PHOTOSYNTHESIS RESEARCH, 91(2-3) (2-3), 232 - 232, English

  Summary international conference


• Evolutionary potential of RuBisCO-like protein in Bacillus subtilis: Interaction with transition-state analogue of RuBisCO.

  Y. Saito, H. Ashida, A. Sekowska, A. Danchin

  SPRINGER, Feb. 2007, PHOTOSYNTHESIS RESEARCH, 91(2-3) (2-3), 231 - 232, English

  Summary international conference


• Isolation and characterization of genes necessary for achievement of RuBisCO accumulation in Arabidopsis thaliana.

  T. Ogawa, K. Nishimura, H. Ashida, A. Yokota

  SPRINGER, Feb. 2007, PHOTOSYNTHESIS RESEARCH, 91(2-3) (2-3), 264 - 264, English

  Summary international conference


• The completion of the Calvin cycle via molecular evolution

  ASHIDA Hiroki, YOKOTA Akiho

  01 Dec. 2006, Journal of plant research, 119, 13 - 13, English


• RuBisCO生合成機構の分子遺伝学的解析

  小川太郎, 蘆田弘樹, 高瀬尚文, 富澤健一, 横田明穂

  2006, 日本植物生理学会年会要旨集, 47th


• Structure-function relationship between photosynthetic RuBisCO and the RuBisCO-like protein of Bacillus subtilis

  Y Saito, S Agnieszka, H Ashida, D Antoine, A Yokota

  OXFORD UNIV PRESS, 2005, PLANT AND CELL PHYSIOLOGY, 46, S75 - S75, English

  Summary international conference


• 光合成CO2固定酵素RuBisCOの起源が明らかに! 枯草菌のメチオニン代謝酵素ＲＬＰに秘められた進化の跡

  蘆田 弘樹

  日本農芸化学会, Jul. 2004, 化学と生物, 42(7) (7), 424 - 426, Japanese

  [Refereed][Invited]


• The common reaction between photosynthetic Ribulosebisphosphate carboxylase/oxygenase (RuBisCO) and Bacillus subtilis RuBisCO-like protein (RLP)

  H Ashida, Y Saitou, K Kobayashi, CJ Kozima, N Ogasawara, A Yokota

  OXFORD UNIV PRESS, 2003, PLANT AND CELL PHYSIOLOGY, 44, S61 - S61, English

  Summary international conference

• 藻類オイル開発研究の最前線―微細藻類由来バイオ燃料の生産技術研究

  岡田 茂, 河野 重行, 神田 英輝, 石川 孝博, 田茂井 政宏, 鈴木 健吾, 社ユーグレナ, 重岡 成, 中嶋 信美, 増田 篤稔, ヤ, 蘆田 弘樹, 広瀬 侑, 池内 昌彦, 関口 弘志

  エヌティーエス, Nov. 2013, ISBN: 4864690855

  


• 形質転換プロトコール 植物編

  田部井 豊

  化学同人, Sep. 2012, ISBN: 475981485X

  


• プラントミメティックス―植物に学ぶ (アドバンスト・バイオミメティックスシリーズ (1))

  甲斐 昌一, 森川 弘道, 鈴木 泰博

  エヌ・ティー・エス, Aug. 2006, ISBN: 4860431316

  


• 広川 タンパク質化学〈4巻〉酵素 4.4 リアーゼ2 (廣川タンパク質化学 4)

  泉井 桂

  廣川書店, Feb. 2005, ISBN: 4567160460

  

• ラパザの核ゲノムコード「盗」葉緑体タンパク質

  丸山萌, 加賀本剛, 大沼亮, 宮城島進也, 蘆田弘樹, 稲田のりこ, 中澤昌美, 粟井光一郎, 柏山祐一郎

  日本共生生物学会第7回大会, Japanese

  Oral presentation


• 植物を用いた未来型医薬用タンパク質生産

  ASHIDA HIROKI

  環境変動の生態・生理学に関する研究会, 2016, Japanese, Domestic conference

  Public discourse


• 光合成CO2固定酵素RuBisCOの機能進化研究からのCO2資源化への展開

  河野 卓成, ASHIDA HIROKI

  日本化学会第９６回春季年会特別企画 生命化学研究から見たCO2資源化：光合成研究と人工光合成の融合を目指して, 2016, Japanese, Domestic conference

  [Invited]

  Invited oral presentation


• C4光合成の炭酸固定酵素と脱炭酸酵素のC3植物への導入による代謝系の改変：水ストレス耐性の向上とその基盤のメタボローム解析

  西村 隆秀, 高木 祐子, 傳寳 雄大, 福崎 英一郎, ASHIDA HIROKI, 陀安 一郎, 秋田 求, 泉井 桂

  日本農芸化学会2016年度大会, 2016, Japanese, Domestic conference

  Poster presentation


• 光合成炭素固定酵素の機能進化

  ASHIDA HIROKI

  環境変動の生態・生理学に関する研究会, Mar. 2015, Japanese, Domestic conference

  Oral presentation


• 光合成CO2固定酵素RuBisCOの機能進化研究

  ASHIDA HIROKI

  日本農芸化学会 農芸化学奨励賞受賞講演会, Mar. 2015, Japanese, Domestic conference

  [Invited]

  Invited oral presentation


• 光合成CO2固定酵素RuBisCOの機能進化研究

  ASHIDA HIROKI

  日本農芸化学会関西支部第492回講演会 2015年度日本農芸化学会奨励賞受賞講演, 2015, Japanese, Domestic conference

  [Invited]

  Invited oral presentation


• ヒトチオレドキシン-1高発現レタス長期食餌負荷による糖尿病モデルマウス血糖コントロールの改善

  渡邉理江, ASHIDA HIROKI, 増谷弘, 三浦ー小林美樹子, 橫田明穂, 淀井淳司

  第３８回 日本分子生物学会, 2015, Japanese, Domestic conference

  Poster presentation


• ‘C4化’タバコにおける水利用効率および乾燥ストレス耐性の向上：δ13Cの測定およびメタボローム解析とアミノ酸分析による性格付け

  泉井 桂, 西村 隆秀, 高木 祐子, 明渡 絵里朱, 秋田 求, ASHIDA HIROKI, 横田 明穂, 陀安 一郎, 中山 泰宗, 傳寶 雄大, 平野 博人, 三輪 哲也

  日本植物生理学会2014年度大会, Mar. 2014, Japanese, Domestic conference

  Oral presentation


• ジャガイモ塊茎を形成するストロンの原基である地中腋芽におけるRanGTPase1遺伝子の発現機構

  加藤 彰, 福井由記, ASHIDA HIROKI, 明石 欣也, 梶川 昌孝, 重岡 成, 橫田 明穂

  日本農芸化学会2014年度大会, Mar. 2014, Japanese, Domestic conference

  Oral presentation


• シアノバクテリアにおけるRuBisCO発現量によるカルボキシソーム形成制御機構

  向川 佳子, 横田 明穂, ASHIDA HIROKI

  日本農芸化学会2014年度大会, Mar. 2014, Japanese, Domestic conference

  Oral presentation


• アーキア型phosphoribulokinaseの機能解析

  河野 卓成, 遠藤 千夏子, 木津 奈津子, 木村 浩之, 溝端 栄一, 井上 豪, 松村 浩由, 横田 明穂, ASHIDA HIROKI

  日本農芸化学会2014年度大会, Mar. 2014, Japanese, Domestic conference

  Oral presentation


• Applications of sequence-specific DNA binding adaptors for assembling proteins on DNA origami

  Huyen T.T. Dinh, Eiji Nakata, Tien A. Ngo, ASHIDA HIROKI, Akiho Yokota, Takashi Morii

  日本化学会第94回春季年会, Mar. 2014, English, 名古屋大学, Domestic conference

  Oral presentation


• バイオ燃料高生産のための炭素固定能を強化したスーパーシアノバクテリアの創成

  ASHIDA HIROKI

  JSTさきがけ第6回領域会議, Feb. 2014, Japanese, Domestic conference

  Others


• 光合成CO2固定酵素ルビスコの機能進化を探る

  ASHIDA HIROKI

  第27回インターゲノミクスセミナー, Dec. 2013, Japanese, Domestic conference

  Public discourse


• バイオ燃料高生産のための炭素固定能を強化したスーパーシアノバクテリアの創成

  ASHIDA HIROKI

  JSTさきがけ研究報告会, Nov. 2013, Japanese, Domestic conference

  Public discourse


• ラン藻を用いたエタノール高生産を目指したルビスコ機能強化研究

  ASHIDA HIROKI

  第4回藻類バイオ燃料生産技術研究会, Sep. 2013, Japanese, Domestic conference

  Public discourse


• 光合成CO2固定酵素ルビスコの基礎研究とその成果を用いた応用研究

  ASHIDA HIROKI

  東京大学生産技術研究所 第1回応用化学セミナー, Jul. 2013, Japanese, Domestic conference

  Public discourse


• アーキアが有する光合成カルビンサイクル酵素phosphoribulokinaseホモログの酵素学的解析

  河野 卓成, 遠藤 千夏子, 横田 明穂, ASHIDA HIROKI

  日本Archaea研究会第26回講演会, Jul. 2013, Japanese, Domestic conference

  Oral presentation


• Enzymatic analysis of archaeal homologues of phosphoribulokinase, a key enzyme in the photosynthetic Calvin cycle.

  Takunari Kohno, Chikako Endo, Akiho Yokota, ASHIDA HIROKI

  30th of the Gordon conference of Archaea, Jul. 2013, English, Italy, International conference

  Poster presentation


• C4光合成の炭素固定酵素（PEPC）と脱炭酸酵素（PCK）を葉緑体内で過剰発現させたタバコ（C3植物）における光合成能、水利用効率（WUE）および浸透圧ストレス耐性

  泉井 桂, 明渡 絵里朱, 西村 隆秀, ASHIDA HIROKI, 橋詰 恵丞, 高木 祐子, 横田 明穂, 秋田 求, 陀安 一郎

  第4回日本光合成学会年会, Jun. 2013, Japanese, Domestic conference

  Oral presentation

• Analysis of the molecular evolution of photosynthetic CO2-fixing pathway

  Ashida Hiroki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Challenging Research (Exploratory), Challenging Research (Exploratory), Kobe University, 28 Jun. 2019 - 31 Mar. 2022

  The Calvin cycle is a primary metabolic pathway for CO2-fixing in plants, algae, and cyanobacteria. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the CO2-fixing enzyme in the Calvin cycle. Methanogenic archaea are not photosynthetic organisms but possess a unique CO2-fixing cycle which is composed of partial steps including RuBisCO catalyzing step from the Calvin cycle and Ribulose monophosphate pathway. Therefore, the methanogenic archaeal CO2-fixing pathway is thought to be a primitive metabolism of the photosynthetic Calvin cycle. In order to study molecular evolution of the Calvin cycle, we analyzed enzymatic properties of RuBisCO in this CO2-fixing pathway. Methanogenic archaeal RuBisCO shows very low maximum reaction rate for the carboxylase reaction and CO2/O2 relative specificity factor, as compared with photosynthetic RubisCOs.


• Improvement of photosynthesis by functional optimization of RuBisCO

  Ashida Hiroki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 01 Apr. 2017 - 31 Mar. 2021, Principal investigator

  We found that the small subunit of Ribulose 1,5-bisphospate carboxylase/oxygenase (RuBisCO) is involved in the determination of CO2/O2 relative specificity in cyanobacterial RuBisCOs. Enzymatic analysis of metanogenic archaeal RuBisCO revealed that this enzyme showed the most lowest CO2/O2 relative specificity among RuBisCOs. We identified RuBisCO activase which catalyzes the activation of this enzyme in cyanobacteria.

  Competitive research funding


• Limit break of photosynthesis by improvement of RuBisCO functions

  Ashida Hiroki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 01 Apr. 2014 - 31 Mar. 2017, Principal investigator

  We found methanogenic archaeal RuBisCO functions in the novel CO2 fixation pathway which has some different steps from the photosynthetic Calvin cycle, suggesting that this novel pathway should be the ancient Calvin cycle. Enzymatic analysis of hybrid RuBisCO created by swap of large and small subunits between thermophilic cyanobacteria RuBisCO with high CO2 specificity and mesophilic cyanobacteria RuBisCO revealed that small subunit was involved in CO2 specificity. Transplastomic tobacco with over expression of RuBisCO activase tends to be enhanced a photosynthetic CO2 fixation rate.

  Competitive research funding


• さきがけ「バイオ燃料高生産のための炭素固定能を強化したスーパーシアノバクテリアの創成」

  蘆田 弘樹

  戦略的創造研究推進事業 個人型研究さきがけ, 2013, Principal investigator

  Competitive research funding


• Molecular basis of productivity improvement through sink development of potato

  YOKOTA Akiho, AKASHI Kinya, ASHIDA Hiroki, MUNEKAGE Yuri

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Nara Institute of Science and Technology, 2009 - 2011

  This study was done to clarify the mechanism of promotion of photosynthesis in the source organ and accumulation of starch in the sink organ in potato plants. The gene was a member of the RanGTPase family. We established the in vitro stolon induction system using potato expalnts with axillary buds to analyze the expression of two RanGTPase genes. The results showed clearly that one of the two endogenous genes was exclusively induced in the initial phase of the stolon induction.


• Enhancement of plant photosynthesis by functional improvement of CO_2-fixing enzyme RuBisCO.

  ASHIDA Hiroki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A), Grant-in-Aid for Young Scientists (A), Nara Institute of Science and Technology, 2009 - 2011

  Residues for functional improvement of RuBisCO were identified by the results form studies of thermophilic cyanobacterial RuBisCO and RuBisCO ancestral protein from Bacillus. I found a pre-Calvin cycle for CO_2 fixation using RuBisCO in methanogenic archaeon. Improvement of plant RuBisCO by replacement with red-algal residue enabled to perform a normal photosynthetic CO_2-fixing with low RuBisCO level in tobacco. The genes necessary for the achievement of RuBisCO accumulation were identified for quantitative enhancement of RuBisCO to increase photosynthetic capacity.


• Improvement of CO2-fixing enzyme, RuBisCO, based on molecular evolution to enhance photosynthesis of plant.

  ASHIDA Hiroki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A), Grant-in-Aid for Young Scientists (A), Nara Institute of Science and Technology, 2006 - 2008

  ルビスコ祖先タンパク質とルビスコの比較解析から、両酵素で共通な触媒残基を明らかにした。これを基に祖先タンパク質にルビスコ触媒必須残基を導入し、試験管内でルビスコへの分子進化に成功した。紅藻ルビスコが持つ高CO_2識別残基をラン藻ルビスコに導入し、CO_2識別能を20%高めることに成功した。さらに、この残基をタバコルビスコに導入したタバコ形質転換体を作出した。紅藻ルビスコ遺伝子導入タバコの解析から、高機能外来性ルビスコの葉緑体機能発現の問題点を明らかにし、その解決策を導き出した。


• Molecular mechanism of functions of photosynthetic CO_2-fixing enzyme RuBisCO

  YOKOTA Akiho, AKASHI Kinya, ASHIDA Hiroki, MUNEKAGE Yuri

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A), Grant-in-Aid for Scientific Research (A), Nara Institute of Science and Technology, 2005 - 2008

  ルビスコの反応触媒の分子機構およびルビスコ生合成の制御機構の解明を行ってきた.ルビスコ反応触媒の分子機構の解明を目指し、すでに枯草菌に発見しているルビスコ活性を持たないルビスコ祖先蛋白質とルビスコの反応中心残基の触媒反応への関わり方を明らかにした.我々が発見したもっとも高いSrel値を持つ紅藻Galdieriaルビスコと植物ルビスコの蛋白質構造比較から、オキシゲナーゼ反応抑制残基と想定している残基をラン藻ルビスコへ導入し、その機能を明らかにした.ルビスコ生合成の制御機構に関しては、ルビスコを正常に合成できない変異株を多数スクリーニングし、分子遺伝学手法によってそれらの変異原因遺伝子を特定し、その機能解析からルビスコ生合成の制御機構を明らかにした.


• 分子進化に基づいたRuBisCOの改良

  補助金, 2005

  光合成カルビンサイクルにおいてCO2固定反応を触媒しているのが、ribulose bisphosphate carbaxylase/oxygenase (RuBisCO)である。RuBisCOはCO2とだけでなくO2との反応（オキシゲナーゼ反応）を示し、オキシゲナーゼ反応はカルボキシラーゼ反応を拮抗的に阻害する。また、RuBisCOの反応速度は一般の酵素と比較し、非常に遅く、その速度は1秒間に２〜３回である。これらの理由から、植物光合成はRuBisCOによって律速されている。このような現状から、O2との反応性を抑制し、反応速度を高めたRuBisCOを創成し、植物内で機能させることによる光合成CO2固定の促進が期待されている。私の研究テーマは、理想型のRuBisCOへの機能改良である。

  Competitive research funding


• 植物光合成改良・光合成CO2固定酵素改良

  ライフサイエンス基礎科学研究, 2005

  光合成CO2固定酵素の改良を行い、植物光合成効率の向上を目指す

  Competitive research funding