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 chr..., , , # Ancient gene clusters govern the initiation of monoterpenoid indole alkaloid biosynthesis and C3 stereochemistry inversion

This dataset contains supplementary files associated with the analyses presented in the manuscript. The data include genome annotations, molecular modeling coordinate files, and structural docking resources that support the identification and characterization of biosynthetic gene clusters and stereochemistry-inverting enzymes involved in monoterpenoid indole alkaloid (MIA) biosynthesis.

## Description of the data and file structure

The dataset is organized into individual compressed archives. Each archive corresponds to a specific analysis described in the manuscript.

* **Rauvolfia_tetraphylla.gff3.zip**
Contains the genome annotation file (`.gff3`) for *Rauvolfia tetraphylla*, generated with **GeMoMa** (reference-based annotation modeler). The annotation is aligned to the *R. tetraphylla* genome assembly (NCBI accession: ASM3051222v1).
* **CrTHAS2_for_XYZ.z...,