Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

Ramakrishnan Krithika,Bajaj Rani,Prabhakar Lal Srivastava,Swati P Kolet,Manojkumar Chopade,Hirekodathakallu V Thulasiram,Mantri Soniya

doi:10.1038/srep08258

Abstract

Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

Highlights

Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value
These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis
We have performed RNA sequencing on Illumina GAII Analyzer, and have screened for unigenes involved in the biosynthesis of secologanin in Catharanthus roseus

Summary

Results and Discussion

The recently reported 8-HGO, which encodes the NADP1 dependent oxidoreductase from C. roseus carries out the dehydrogenation of 10-hydroxygeraniol and other acyclic monoterpenes[19], but does not possess much sequence similarity with Cr10HGO In contrast to these observations, monohydroxy terpene derivatives such as geraniol, nerol, and farnesol were found to be poor substrates for Cr10HGO as compared to the reported 8HGO19 (Supplementary Table S2 and Supplementary Fig. S4). CrG10H was cloned using the primers designed from the reported gene, which encodes geraniol hydroxylase in C. roseus[23] as the unigenes 742 had similar sequence to that of the reported one (Supplementary Table S4) These genes were cloned in various expression vectors compatible with E. coli or yeast systems and the expressed proteins were purified by Ni-NTA chromatography, except CrG10H, which remained as microsomal pellet when expressed in yeast system (Supplementary Figures S19–S27). Further we have demonstrated the in vitro formation of (1R, 4aS, 7S, 7aR)nepetalactol when geraniol was incubated with CrG10H, Cr10HGO and CrIDS

Methods

Author contributions

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