title: Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis.
abstract: Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
authors.forename: Akiyuki
authors.initials: A
authors.lastname: Takaya
authors.forename: Yuan-Wei
authors.initials: YW
authors.lastname: Zhang
authors.forename: Kasem
authors.initials: K
authors.lastname: Asawatreratanakul
authors.forename: Dhirayos
authors.initials: D
authors.lastname: Wititsuwannakul
authors.forename: Rapepun
authors.initials: R
authors.lastname: Wititsuwannakul
authors.forename: Seiji
authors.initials: S
authors.lastname: Takahashi
authors.forename: Tanetoshi
authors.initials: T
authors.lastname: Koyama
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D051231:Farnesyltranstransferase; D035264:Flowers / Q000378:metabolism; D015723:Gene Library; D028482:Hevea / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007840:Latex / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D018515:Plant Leaves / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D007840:Latex; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase