title: Molecular cloning and biochemical characterization of isoprene synthases from the tropical trees Ficus virgata, Ficus septica, and Casuarina equisetifolia.
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 Mg(2+), whereas IspS from C. equisetifolia was dependent more on Mn(2+) than on Mg(2+). 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.
authors.affiliation: Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan, okuhiros@comb.u-ryukyu.ac.jp.
authors.forename: Hirosuke
authors.initials: H
authors.lastname: Oku
authors.forename: Masashi
authors.initials: M
authors.lastname: Inafuku
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Tnomonori
authors.initials: T
authors.lastname: Takamine
authors.forename: Mutanda
authors.initials: M
authors.lastname: Ishmael
authors.forename: Masakazu
authors.initials: M
authors.lastname: Fukuta
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D030681:Ficus / Q000235:genetics / Q000378:metabolism; D007564:Japan; D019684:Magnoliopsida / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D012333:RNA, Messenger; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase