Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis

Akiyuki Takaya, Yuan Wei Zhang, Kasem Asawatreratanakul, Dhirayos Wititsuwannakul, Rapepun Wititsuwannakul, Seiji Takahashi, Tanetoshi Koyama

Research output: Contribution to journalArticlepeer-review

50 Citations (Scopus)

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 Km values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni2+-affinity column were 24.1, 6.8, 2.3, and 11.5 μM, respectively. The enzyme shows optimum activity at approximately 40°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.

Original languageEnglish
Pages (from-to)214-220
Number of pages7
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1625
Issue number2
DOIs
Publication statusPublished - 2003 Jan 27

Keywords

  • Geranylgeranyl diphosphate
  • Hevea brasiliensis
  • Isoprenoid
  • Prenyl chain elongation
  • Prenyltransferase
  • Rubber biosynthesis

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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