Abstract
Linear prenyl diphosphates whose carbon chain length varies widely from geranyl diphosphate (C10) to natural rubber (C>105) are biosynthesized by the catalytic function of a group of enzymes called prenyltransferases. Prenyltransferases are classified in two major groups, trans- or (E)-prenyltransferases and cis- or (Z)-prenyltransferases. From the year of 1987, many genes encoding (E)-prenyltransferases have been cloned and characterized well. However, the structure and detailed mechanism of (Z)-prenyltransferase had been completely unknown until our identification of a gene encoding the undecaprenyl diphosphate synthase (UPS) from Micrococcus luteus B-P 26. Not only the primary structure but also the tertiary structure of the UPS is quite different from those of (E)-prenyltransferases. Multiple alignment of primary structures of (Z)-prenyltransferases identified from various organisms reveals that there are five highly conserved regions among (Z)-prenyltransferases. In order to elucidate the biosynthetic pathway of natural rubber which is composed of cis-1, 4-polyisoprene, we isolated and characterized two genes encoding (Z)-prenyltransferases from the latex in Hevea brasiliensis by using sequence information on the conserved regions of (Z)-prenyltransferases. In vitro rubber transferase assay using the recombinant gene product revealed that the enzyme catalyzed the formation of polyprenyl products with approximate sizes around 1×104 Da. Moreover, in the presence of washed bottom fraction from latex, the rubber transferase activity and the size of the major product was increased, suggesting a possible requirement of certain activation factors in the washed bottom fraction for the production of high molecular weight rubber.
