Abstract
Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5 beta-reductase (P5 beta R) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5 beta R genes. Characterization of recombinant CrP5 beta R proteins demonstrates that all but CrP5 beta R3 can reduce progesterone and thus can be classified as P5 beta Rs. Three of them, namely CrP5 beta R1, CrP5 beta R2, and CrP5 beta R4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5 beta R5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5 beta R4 may also participate in secoiridoid biosynthesis. We cloned a set of P5 beta R genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5 beta R proteins and has evolved early during evolution.
