Ancient gene clusters govern the initiation of monoterpenoid indole alkaloid biosynthesis and C3 stereochemistry inversion

The inversion of C3 stereochemistry in monoterpenoid indole alkaloids
(MIAs), derived from the central precursor strictosidine (3S), is a
critical step for the biosynthesis of numerous 3R MIAs and spirooxindoles,
including the antihypertensive drug reserpine. While early MIA
biosynthesis preserves the 3S configuration, the mechanism underlying C3
inversion has remained unresolved. Here, we identify and biochemically
characterize a conserved oxidase-reductase pair in the Gentianales order:
the heteroyohimbine/yohimbine/corynanthe C3-oxidase (HYC3O) and
C3-reductase (HYC3R), which together invert the 3S stereochemistry to 3R
across diverse substrates. Notably, HYC3O and HYC3R reside in gene
clusters in Rauvolfia tetraphylla and Catharanthus roseus, homologous to
an elusive geissoschizine synthase (GS) cluster we also uncovered. In R.
tetraphylla, these clusters are in tandem on a single chromosome, likely
derived from segmental duplication, whereas in C. roseus they reside on
separate chromosomes due to translocation. Comparative genomics indicate
the GS cluster originated at the base of Gentianales (∼135 Mya),
coinciding with the evolution of the strictosidine synthase cluster, while
the reserpine cluster arose later in rauvolfioid Apocynaceae. Together,
these findings uncover the genomic and biochemical basis for key events in
MIA evolution and diversification, providing insights beyond the canonical
vinblastine and ajmaline biosynthetic pathways.