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Báo cáo khoa học: Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis A key factor participating in natural rubber biosynthesis

Natural rubber fromHevea brasiliensisis a high molecular mass polymer of isoprene units withcis-configuration. The enzyme responsible for the cis-1,4-polymerization of iso-prene units has been identified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have iso-lated and characterized cDNAs from H. | Eur. J. Biochem. 270 4671-4680 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03863.x Molecular cloning expression and characterization of cDNA encoding c s-prenyltransferases from Hevea brasiliensis A key factor participating in natural rubber biosynthesis Kasem Asawatreratanakul1 2 Yuan-Wei Zhang1 . Dhiravos Wititsuwannakul3 Rapepun Wititsuwannakul4 Seiji Takahashi1 Atiya Rattanapittayaporn4 and Tanetoshi Koyama1 Institute of Multidisciplinary Research for Advanced Materials Tohoku University Sendai Japan department of Chemistry Thaksin University Songkla Thailand 3Department of Biochemistry Mahidol University Bangkok Thailand department of Biochemistry Prince of Songkla University Hat-Yai Thailand Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration. The enzyme responsible for the cis-1 4-polymerization of isoprene units has been identified as a particle-bound rubber transferase but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase. Northern blot analysis indicated that the transcript s of the Hevea cis-prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 X 103-1 X 104 Da. Moreover in the presence of washed bottom fraction particles from latex the rubber transferase activity producing .