A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids

Abstract

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to g-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 g-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the g-tocopherol-like N-methyltransferase family from g-tocopherol methyltransferases.

Isolation and Characterization of Reticuline N-Methyltransferase Involved in Biosynthesis of the Aporphine Alkaloid Magnoflorine in Opium Poppy

“…Unlike O-methyltransferases, which are considered monophyletic in origin (23), plant NMTs were clearly recruited independently from genes encoding several unrelated ancestral enzymes. Major and distinct clades are represented in plants by (i) putrescine NMTs (24), (ii) phosphoethanolamine NMTs (25), (iii) xanthine NMTs (26,27), (iv) enzymes related to tocopherol C-methyltransferases and involved in monoterpenoid indole alkaloid biosynthesis (28,29), and (v) NMTs involved in BIA metabolism (13,15,16). Most plant NMTs carry out a single methylation of the acceptor nitrogen atom; however, sequential methylation is a common feature of phosphoethanolamine NMTs, which yield the quaternary ammonium betaines used as osmolytes under certain stress condi- tions.…”

Rauvolfia serpentina N‐methyltransferases involved in ajmaline and Nβ‐methylajmaline biosynthesis belong to a gene family derived from γ‐tocopherol C‐methyltransferase

“…While phylogenetic analysis (Figure S3, Table S3) clearly associated RSNNMT and RSANMT with the tocopherol C ‐methytransferases rather than with tabersonine‐16‐ O ‐methytransferase, these genes segregated with other TLMTs rather than with TMTs. This corroborates previous larger phylogenetic analyses (Liscombe et al ., ; Levac et al ., ) showing that TLMTs can be distinguished from TMTs and that they may have evolved from TMTs to perform N ‐methylations of MIAs.…”

“…Recently, a previously uncharacterized family of AdoMet‐dependent N ‐methyltransferases phylogenetically related to the γ‐tocopherol C ‐methyltransferases was identified in several members of the Apocynaceae (Liscombe et al ., ; Levac et al ., ). This family of γ‐tocopherol‐like N ‐methyltransferases (γ‐TLMTs) catalyzes N ‐methylation in MIA biosynthesis.…”

“…This led to the identification of five γ‐TLMT candidate genes that might be responsible for the final methylation step in ajmaline biosynthesis. This approach resulted in the molecular and biochemical characterization of picrinine N ‐methyltransferases from R. serpentina and from Vinca minor , respectively (Levac et al ., ). The present study describes the molecular and biochemical characterization of two other γ‐TLMT genes from R. serpentina , norajmaline N ‐methyltransferase (RsNNMT) that indole N ‐methylates norajmaline and ajmaline N β ‐methyltransferase (RsANMT) that N β ‐methylates ajmaline (Figure ).…”

Vitamin E

“…The vitamin E biosynthetic pathway has been well elucidated in model species, and select genes have been transformed and overexpressed individually or collectively in various plants to improve vitamin E content and composition [26,87,125,128,[140][141][142][143][144][145][146][147][148][149]. For oil crops, the enhancement of γ-tocopherol is important in order to prevent oil peroxidation and thus to improve oil quality [150].…”