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Molecular cloning and biochemical characterization of isoprene synthases from the tropical trees Ficus virgata, Ficus septica, and Casuarina equisetifolia

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Abstract

Three isoprene synthase (IspS) cDNA clones have been isolated from tropical trees (Ficus septica, F. virgata, and Casuarina equisetifolia), and their enzyme properties have been compared with those of Populus alba IspS. Phylogenetic analysis of the deduced amino acid sequences with known monoterpene synthase resolved IspS from F. septica and F. virgata and other IspSs in a clade together with TPS-b clade I, whereas IspS from C. equisetifolia was within another clade, sister to TPS-b clade II. The optimum reaction temperature was 40 °C for the IspSs isolated from the tropical trees, and 45 °C for P. alba IspS. The optimum pH of the IspSs from the tropical trees peaked between pH 8 and pH10 contrasting with the rather broad optimum pH (7.5–10.5) of P. alba IspS. IspSs from F. septica and F. virgata were activated solely by Mg2+, whereas IspS from C. equisetifolia was dependent more on Mn2+ than on Mg2+. Michaelis constant (Km) values of IspSs from tropical trees were lower than that of P. alba IspS. Analysis of inter fragment interaction energy of IspS-substrate complex model and crystal structure of bornyl diphosphate synthase (1N20) found that the coordination geometry of amino acids with higher attraction force is similar at the active site of C. equisetifolia IspS and bornyl diphosphate synthase. These observations suggest the occurrence of another group of IspSs in TPS-b subfamily and extend the knowledge on biochemical regulatory mechanism of isoprene emission from tropical trees.

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Acknowledgments

The authors thank Professor Yazaki, Kyoto University for the generous provision of the P. alba IspS cDNA. The authors also thank Dr. Ryo Yanagitta for his help with the use of Autodock program and Dr. Seikoh Saitoh for the installation and operation of PAICS program. Our thanks also go to Mr. Motoi Itoh, Naeko Miyazato, Narumi Tsunoda, Shin Kedashiro for their contribution in the cloning of IspS cDNA.

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Authors and Affiliations

  1. Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan

    Hirosuke Oku & Masashi Inafuku

  2. Faculty of Agriculture, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan

    Tnomonori Takamine & Masakazu Fukuta

  3. United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan

    Mutanda Ishmael

  4. Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, 852-8521, Japan

    Takeshi Ishikawa

  5. Molecular Biotechnology Group, Center of Molecular Bioscience, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan

    Hirosuke Oku

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  1. Hirosuke Oku
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  2. Masashi Inafuku
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  3. Takeshi Ishikawa
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Correspondence to Hirosuke Oku.

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Oku, H., Inafuku, M., Ishikawa, T. et al. Molecular cloning and biochemical characterization of isoprene synthases from the tropical trees Ficus virgata, Ficus septica, and Casuarina equisetifolia . J Plant Res 128, 849–861 (2015). https://doi.org/10.1007/s10265-015-0740-9

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  • DOI: https://doi.org/10.1007/s10265-015-0740-9

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