Alkaloid Biosynthesis in Rauvolfia- cDNA Cloning of Major Enzymes of the Ajmaline Pathway

All the major reactions taking part in the biosynthesis of ajmaline in cell suspension cultures of the Indian medicinal plant Rauvolfia have now been established at the enzyme level. Of the well investigated 10 steps six of the involved enzymes have been functionally cloned. These are strictosidine synthase (STR), strictosidine glucosidase (SG), polyneuridine aldehyde esterase (PNAE), vinorine synthase (VS), cytochrome P450 reductase (CPR) and acetylajmalan acetylesterase (AAE). Because the cDNAs of these enzymes are now known their detailed molecular analysis became attainable for the first time. Some of these enzymes such as STR, SG or VS could be produced in E. coli at a preparative scale resulting in mg amounts of pure enzymes. They also have been crystallized and their preliminary X-ray analyses were published recently. It is only a matter of time that their molecular structure and the mechanisms of the catalyzed reactions will be elucidated. Of the soluble enzymes vomilenine reductase and 1.2-dihydrovomilenine reductase remain to be heterologously expressed. Appropriate cDNA clones have recently been isolated. What membrane bound enzymes of this pathway is concerned cloning could not be achieved up to now. Our future strategy is to purify these enzymes first and to use the "reverse genetic" approach as we did for the soluble enzymes. The sarpagine bridge enzyme (SBE), vinorine hydroxylase (VH) and norajmaline N-methyltransferase (NAMT) are the only enzymes which remain as the major candidates for expression studies in order to express heterologously the complete ajmaline biosynthetic pathway.