A novel cinnamyl alcohol dehydrogenase (CAD)-like reductase contributes to the structural diversity of monoterpenoid indole alkaloids in Rauvolfia

Abstract

Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites.

Overexpression of Artemisia annua Cinnamyl Alcohol Dehydrogenase Increases Lignin and Coumarin and Reduces Artemisinin and Other Sesquiterpenes

“…Accordingly, it is likely that overexpression of AaCAD ( Figure 3E ) enhances the catalytic conversion of artemisinin aldehyde to alcohol in the presence of NADPH in trichomes. In addition to our observations, CAD or CAD-like enzymes have been reported to be involved in the formation of monoterpenoid indole alkaloids in Rauvolfia serpentine ( Geissler et al, 2016 ). Moreover, additional biochemical studies have shown that CAD is characterized by a high promiscuity level of different substrates ( Chao et al, 2014 ; Geissler et al, 2016 ).…”

“…In addition to our observations, CAD or CAD-like enzymes have been reported to be involved in the formation of monoterpenoid indole alkaloids in Rauvolfia serpentine ( Geissler et al, 2016 ). Moreover, additional biochemical studies have shown that CAD is characterized by a high promiscuity level of different substrates ( Chao et al, 2014 ; Geissler et al, 2016 ). Based on our and other reports, one hypothesis is that CAD-mediated crosstalk can occur in other metabolic pathways.…”

An integrative overview of physiological and proteomic changes of cytokinin‐induced potato (Solanum tuberosum L.) tuber development in vitro

“…The upregulation of FDH (spot 6503) with the increase of exogenous BAP concentration might accelerate one‐carbon metabolism during tuber development. Perakine reductase‐like (spot 3408) and 8‐hydroxy geraniol dehydrogenase‐like (spots 4519 and 5504), participating in the biosynthesis of monoterpenoid indole alkaloids (Sun et al , Miettinen et al , Geissler et al ), were also upregulated with the increase of exogenous BAP concentrations. NADH‐cytochrome b5 reductase (CBR) serves as electron donor for cytochrome b5 (a ubiquitous electron carrier), thus participating in a variety of metabolic pathways.…”

Dual Catalytic Activity of a Cytochrome P450 Controls Bifurcation at a Metabolic Branch Point of Alkaloid Biosynthesis in Rauwolfia serpentina

“…Like all MIAs, ajmaline is produced via the versatile intermediate strictosidine, which is subject to multiple catalytic steps to yield the structurally diverse members of the MIA natural product family . All biosynthetic transformations leading from strictosidine to ajmaline have been detected in enzyme fractions from Rauwolfia cell culture, though only six biosynthetic genes have been cloned and characterized (Figure ) . Several studies showed that vomilenine is an ajmaline biosynthetic intermediate that is produced through selective hydroxylation of vinorine at the C‐21 position by Rauwolfia cell culture extracts (Figure ) .…”