Two Tabersonine 6,7-Epoxidases Initiate Lochnericine-Derived Alkaloid Biosynthesis in Catharanthus roseus

Abstract

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. and displayed complementary expression profiles, with expressed mainly in roots and in aerial organs. Our results suggest that and originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

The assembly of (+)‐vincadifformine‐ and (−)‐tabersonine‐derived monoterpenoid indole alkaloids in Catharanthus roseus involves separate branch pathways

“…In marked contrast, both (+)‐minovincinine (Figure , inset lane 8) and its O ‐acetylated derivative (+)‐echitovenine (Figure , inset lane 9) accumulated, but not (+)‐vincadifformine (Figure , inset lane 7). While enantiomeric forms of the MIAs were not specified, hairy roots, adventitious roots and roots from whole C. roseus plants (Carqueijeiro et al ., ,b) were reported to accumulate tabersonine and lochnericine together with lower levels of hörhammericine, vincadifformine, minovincinine and echitovenine, while only traces of 19‐hydroxytabersonine, 19‐ O ‐acetyltabersonine or 19‐ O ‐acetylhörhammericine were detected.…”

“…The results obtained with the T3O homolog showed that these two separate enzymes, sharing 27% amino acid identity, are specific for enantiomer‐specific hydroxylation of (+)‐ and (−)‐aspidosperma skeletons, respectively (Figure ). The results suggest that while 19‐ O ‐acetylhörhammericine is derived from (−)‐tabersonine (Figure ) (Carqueijeiro et al ., ,b), formation of (+)‐echitovenine involved a separate two‐step pathway from (+)‐vincadifformine (Figure ). The T3O homolog that catalyzes this regiospecific and stereospecific hydroxylation was named (+)‐vincadifformine 19‐hydroxylase (V19H).…”

“…It has been suggested (Carqueijeiro et al ., ,b) that TAT rather than MAT (Laflamme et al ., ) catalyzes the terminal reactions for the biosynthesis of 19‐ O ‐acetylhörhammericine and echitovenine (Figure ). When (+)‐minovincinine (Figure j) was incubated with recombinant purified MAT and acetyl‐CoA, (+)‐echitovenine (Figure k) was formed, while TAT was not able to accept this (+)‐enantiomer as a substrate (Figure l).…”

“…Little Bright Eye plants also show that vindolinine and 19 S ‐vindolinine are major MIAs in mature roots (Ferreres et al ., ). While less is known about MIA biosynthesis in C. roseus roots, the pathway for assembly of 19‐ O ‐acetylhörhammericine from tabersonine involves two cytochrome P450‐mediated reactions for 6,7‐epoxide (Rodriguez et al ., ; Carqueijeiro et al ., 2018a) and 19‐hydroxyl (Giddings et al ., ) formation followed by 19‐ O ‐acetylation catalyzed by minovincinine 19‐hydroxy‐ O ‐acetyltransferase (MAT) (Laflamme et al ., ) (Figure 2). However, the recent discovery of another root‐specific 19‐hydroxytabersonine, 19‐ O ‐acetyltransferase (TAT) (Carqueijeiro et al ., 2018b), capable of acetylating both minovincinine and hörhammericine with 200‐fold higher catalytic efficiency than MAT, led to the suggestion that MAT may only play a minor role in O ‐acetylation of these tabersonine and vincadifformine derivatives in C. roseus roots.…”

“…The present study describes the functional characterization of (+)‐vincadifformine 19‐hydroxylase (V19H), a root‐specific cytochrome P450 with high amino acid sequence similarity to tabersonine‐3‐oxygenase (T3O; CYP71D1V1) which is involved in vindoline biosynthesis in C. roseus leaves. The substrate specificity of this CYP for the (+)‐enantiomer of vincadifformine (Figure ) shows that formation of (+)‐echitovenine (Figure ) occurs through a separate enantiomer‐specific pathway from the one recently documented for the formation of the hörhammericine (Laflamme et al ., ; Carqueijeiro et al ., 2018a), another major MIA found in C. roseus roots. The study follows up on the discovery that biosynthesis of vincadifformine branches off prior to the formation of (−)‐tabersonine from a common MIA intermediate (Qu et al ., ) to form the (+)‐enantiomer that is converted to (+)‐19‐hydroxyvincadifformine (or (+)‐minovincinine) by V19H, while V19H is not active when the (−)‐enantiomer of vincadifformine is used as a substrate (Figure ).…”