Search

SUMMARY: Cytochrome P450 monooxygenases (CYPs) are well known for their ability to catalyze diverse oxidation reactions, playing a significant role in the biosynthesis of various natural products. In the realm of monoterpenoid indole alkaloids (MIAs), one of the largest groups of alkaloids in nature, CYPs are integral to reactions such as hydroxylation, epoxidation, ring opening, ring rearrangement, and aromatization, contributing to the extensive diversification of these compounds. In this study, we investigate the transcriptome, metabolome, and MIA biosynthesis in Tabernaemontana litoralis (milky way tree), a prolific producer of rare pseudoaspidosperma‐type MIAs. Alongside known pseudoaspidosperma biosynthetic genes, we identify and characterize three new CYPs that facilitate regio‐ and stereospecific oxidation of four MIA skeletons: iboga, aspidosperma, pseudoaspidosperma, and quebrachamine. Notably, the tabersonine 14,15‐β‐epoxidase catalyzes the formation of pachysiphine, the stereoisomer of 14,15‐α‐epoxytabersonine (lochnericine) found in Catharanthus roseus (Madagascar periwinkle) roots. The pseudovincadifformine 18‐hydroxylase is the first CYP identified to modify a pseudoaspidosperma skeleton. Additionally, we demonstrate that the enzyme responsible for C10‐hydroxylation of the iboga MIA coronaridine also catalyzes C10‐hydroxylation of voaphylline, which bears a quebrachamine skeleton. With the discovery of a new MIA, 11‐hydroxypseudovincadifformine, this study provides a comprehensive understanding of MIA biosynthesis and diversification in T. litoralis, highlighting its potential for further exploration. Significance Statement: New cytochrome P450 monooxygenases (CYP) from Tabernaemontana litoralis can oxidize four different monoterpenoid indole alkaloid (MIA) skeletons, including the rare pseudovincadifformine alkaloid. Our discovery highlights the remarkable versatility of CYP enzymes as catalysts in the biosynthesis of natural products. Such flexibility is a key factor driving the structural diversity of medicinal MIAs, which encompass over 3000 unique structures and hold significant potential for therapeutic development. [ABSTRACT FROM AUTHOR]

Monoterpenoid indole alkaloids (MIA) are one of the largest and most complex alkaloid class in nature, boasting many clinically significant drugs such as anticancer vinblastine and antiarrhythmic ajmaline. Many MIAs undergo nitrogen N-methylation, altering their reactivity and affinity to the biological targets through a straightforward reaction. Remarkably, all known MIA N-methyltransferases (NMT) originate from the neofunctionalization of ancestral γ-tocopherol C-methyltransferases (γTMTs), a phenomenon seemingly unique to the Apocynaceae family. In this study, we unveil and characterize a new γTMT-like enzyme from the plant Tabernaemontana elegans (toad tree): perivine Nγ-methyltransferase (TePeNMT). TePeNMT and other homologs form a distinct clade in our phylogenetic study, setting them apart from other γTMTs and γTMT-like NMTs discovered to date. Enzyme kinetic experiments and enzyme homology modeling studies reveal the significant differences in enzyme active sites between TePeNMT and CrPeNMT, a previously characterized perivine Nβ-methyltransferase from Catharanthus roseus (Madagascar periwinkle). Collectively, our findings suggest that parallel evolution of ancestral γTMTs may be responsible for the occurrence of perivine N-methylation in T. elegans and C. roseus. [ABSTRACT FROM AUTHOR]