Ancient gene clusters govern the initiation of monoterpenoid indole alkaloid biosynthesis and C3 stereochemistry inversion
J. Y. Hwang, Jonathan Kirshner, Daniel André Ramey Deschênes, Matthew Bailey Richardson, Steven J. Fleck, S. G. MANN, Jun Guo, Jacob Owen Perley, Mohammadamin Shahsavarani, Jorge Jonathan Oswaldo Garza‐García, Alyssa Dawn Seveck, Savannah Sadie Doiron, Zhan Mai, Stephen Nelson Silliphant, Sarah A. Englehart, Barry A. Blight, Larry A. Calhoun, Di Gao, Jiazhang Lian, Ghislain Deslongchamps, Victor A. Albert, Yang Qu
Abstract
The inversion of C3 stereochemistry in monoterpenoid indole alkaloids (MIAs), derived from the central precursor strictosidine (3S), is essential for producing pharmacologically important 3 R MIAs and spirooxindoles such as 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 Gentianales: heteroyohimbine/yohimbine/corynanthe C3-oxidase (HYC3O) and C3-reductase (HYC3R), which together invert the 3S stereochemistry to 3 R across diverse substrates. HYC3O and HYC3R are encoded within biosynthetic gene clusters in Rauvolfia tetraphylla and Catharanthus roseus, homologous to a geissoschizine synthase (GS) cluster also uncovered. Comparative genomics indicate that the GS cluster originated at the base of Gentianales (~135 Mya), coinciding with the evolution of the strictosidine synthase cluster, whereas the reserpine cluster arose later. These findings uncover the genomic and biochemical basis of key events driving MIA diversification beyond canonical vinblastine and ajmaline pathways. Inversion of C3 stereochemistry of monoterpenoid indole alkaloids (MIAs) has to occur at some point during their biosynthesis; however, the mechanism has remained unresolved. Here, the authors report an oxidase–reductase enzyme pair encoded within a gene cluster and demonstrate their collaborative role in inverting MIA C3 stereochemistry.