Molecular cloning and characterization of a functional cDNA clone encoding isopentenyl diphosphate isomerase from Hevea brasiliensis

Plants produce many isoprenoid compounds of biological and/or economic importance. The first step in isoprenoid biosynthesis is the isomerization of isopentenyl diphosphate (IPP) to its electrophilic isomer, dimethylallyl diphosphate (DMAPP), by the enzyme IPP isomerase (EC 5.3.3.2). The following successive head-to-tail condensation reactions of the five-carbon intermediates lead to the synthesis of a variety of isoprenoids. Among these polyisoprenes, natural rubber is an important raw material with many industrial uses. In order to build our knowledge base of rubber biosynthesis, we cloned and characterized two cDNA clones encoding the IPP isomerase from the latex of Hevea brasiliensis, where a large quantity of natural rubber (cis-1,4-polyisoprene) is produced. The two clones differ only in the untranslated regions, and have a continuous open reading frame encoding a peptide of 234 amino acids with a predicted molecular mass of 26.7 kDa. The deduced protein is acidic, with an isoelectric point of 4. 7, and shows high sequence identity with other IPP isomerases. The recombinant protein expressed in Escherichia coli showed IPP isomerase activity. In vitro rubber biosynthesis assays using washed rubber particles deprived of initiating allylic diphosphates, revealed that the recombinant IPP isomerase is functional in catalyzing the conversion of IPP to DMAPP, a key activation step of the basic five-carbon isoprene unit in isoprenoid biosynthesis. Southern analysis indicated that the IPP isomerase is encoded by two genes in Hevea rubber tree. Analyses of RNA extracted from extruded latex of the trees, wounded with nails, showed that wounding did not change the transcript level of IPP isomerase.