DOI: 10.5061/dryad.vdncjsz59
title: Ancient gene clusters govern the initiation of monoterpenoid indole alkaloid biosynthesis and C3 stereochemistry inversion

description: 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.
description: # 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.zip** Contains `.xyz` coordinate files for docking
 analyses of **Catharanthus roseus tetrahydroalstonine synthase 2
 (CrTHAS2)** with ligands. Files were generated using **Molecular Operating
 Environment (MOE)**. * **HpHYC3O_for_XYZ.zip** Contains `.xyz` coordinate
 files for **Hameliapatens HYC3O (HpHYC3O)** enzyme with ligands, generated
 in MOE. * **RsHYC3R(CAD7)_for_XYZ.zip** Contains `.xyz` coordinate files
 for **Rauvolfia serpentina C3-reductase (RsHYC3R, also referred to as
 CAD7)** with ligands, generated in MOE. * **RtHYC3O_for_XYZ.zip** Contains
 `.xyz` coordinate files for **Rauvolfia tetraphylla HYC3O (RtHYC3O)**
 enzyme with ligands, generated in MOE. All XYZ files contain Cartesian
 atomic coordinates for enzyme–ligand complexes used in substrate docking
 and structural analyses. These are intended for reuse in molecular
 modeling workflows. ## Variable and file definitions * **.gff3 annotation
 file** * `seqid`: Chromosome or scaffold identifier * `source`: Annotation
 source (GeMoMa) * `type`: Feature type (e.g., gene, mRNA, exon, CDS) *
 `start`, `end`: Genomic coordinates (base pairs) * `score`:
 Alignment/annotation score (NA if not applicable) * `strand`: + / – strand
 * `phase`: CDS phase (0–2; NA if not CDS) * `attributes`: Gene ID,
 transcript ID, functional annotations * **.xyz coordinate files** *
 `ATOM`: List of atoms in the modeled complex * `X, Y, Z`: Cartesian
 coordinates (Ångströms) * `element`: Atomic symbol (C, N, O, H, etc.) *
 Each file corresponds to a specific enzyme–ligand docking pose. ##
 Sharing/Access information * Genome assembly reference: *Rauvolfia
 tetraphylla* genome (ASM3051222v1) is publicly available on NCBI. *
 Experimental context and analyses are described in the associated
 publication. ## Code/Software * **Genome annotation**: Generated with
 **GeMoMa** (v1.9). Open-source software available at:
 [https://github.com/JStrozzi/GeMoMa](https://github.com/JStrozzi/GeMoMa).
 * **Molecular modeling and docking**: Performed using **Molecular
 Operating Environment (MOE), 2022.02 (Chemical Computing Group Inc.)**. *
 **Viewing files**: * `.gff3`: Open with any genome browser or text editor
 (e.g., **IGV**, **Apollo**, **Artemis**, or command-line parsing). *
 `.xyz`: Can be viewed with free/open-source molecular visualization tools,
 such as **PyMOL (open-source edition)**, **Avogadro**, or **VMD**. ##
 Notes for reuse * The `.gff3` annotation is specific to the *R.
 tetraphylla* genome assembly ASM3051222v1 and may not align properly to
 other assemblies. * XYZ coordinate files represent modeled docking poses;
 they are not experimentally determined structures. They can be used as
 starting points for comparative docking, energy minimization, or
 structural refinement. * No additional preprocessing is required to use
 these files.