Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana. Implications in rubber biosynthesis.
cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity. Despite potential significance of cis-prenyltransferase in plant growth and development, no gene of the enzyme has been cloned from higher plants. Using sequence information of the conserved region of cis-prenyltransferase cloned recently from Escherichia coli, Micrococcus luteus, and yeast, we have isolated and characterized the first plant cis-prenyltransferase from Arabidopsis thaliana. Sequence analysis revealed that the protein is highly homologous in several conserved regions to cis-prenyltransferases from M. luteus, E. coli, and yeast. In vitro analyses using the recombinant protein overexpressed in E. coli revealed that the enzyme catalyzed the formation of polyprenyl diphosphates ranging in carbon number from 100 to 130 with a predominance of C(120). The enzyme exhibited a higher affinity for farnesyl diphosphate than for geranylgeranyl diphosphate, with the K(m) values being 0.13 and 3.62 micrometer, respectively, but a lower affinity for isopentenyl diphosphate, with a K(m) value of 23 micrometer. In vitro rubber biosynthesis analysis indicated that the Arabidopsis cis-prenyltransferase itself could not catalyze the formation of higher molecular weight polyprenyl diphosphates similar to natural rubber. A reverse transcriptase-polymerase chain reaction analysis showed that the gene was expressed at low levels in Arabidopsis plant, in which expression of the cis-prenyltransferase in leaf and root was higher than that in stem, flower, and silique. These results indicate the tissue-specific expression of cis-prenyltransferase and suggest a potential role and significance of the enzyme in the polyisoprenoid biosynthesis in plants.
Oh
S K
SK
Kumho Life and Environmental Science Laboratory, 1 Oryong-Dong, Puk-Gu, Kwangju, 500-712, Korea.
Han
K H
KH
Ryu
S B
SB
Kang
H
H
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012408:Rubber; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics / Q000502:physiology*
D017830:Octoxynol; D012408:Rubber; D014166:Transferases; C106821:cis-prenyl transferase; D008274:Magnesium