A single CYP76 enzyme catalyzes the G8H step in Camptotheca acuminata iridoid biosynthesis

The geraniol-derived (seco)iridoid skeleton serves as a fundamental precursor for bioactive monoterpene indole alkaloids (MIAs). Geraniol 8-hydroxylase (G8H), a cytochrome P450 monooxygenase, catalyzes the first committed step of iridoid biosynthesis by hydroxylating geraniol to 8-hydroxygeraniol. This study aims to functionally characterize three homologous CaG8H genes from Camptotheca acuminata and elucidate their distinct roles in iridoid metabolism. Three CaG8H homologs (CaG8H1-3) were identified through phylogenetic and bioinformatic analyses. CaG8H2 was heterologously expressed in Saccharomyces cerevisiae for in vitro enzymatic assays. And in vivo functional verification of the genes using virus-induced gene silencing (VIGS) combined with metabolite quantification by HPLC/GC-MS. Phylogenetic analysis classified CaG8H2 and CaG8H3 as CYP76B subfamily members, while CaG8H1 belongs to CYP76C. Recombinant CaG8H2 specifically converted geraniol to 8-hydroxygeraniol with 23.98 min retention time in GC-MS analysis, whereas CaG8H1 and CaG8H3 showed no detectable activity toward geraniol. VIGS-mediated silencing of CaG8H2 and CaG8H3 reduced their expression by 49% and 57%, respectively, resulting in 41% and 26% decreases in loganic acid and camptothecin accumulation. CaG8H1 silencing had minimal effects on metabolite levels. Among three CYP76 homologs, only CaG8H2 exhibits geraniol 8-hydroxylase activity in vitro, while CaG8H2 and CaG8H3 collectively regulate iridoid biosynthesis in vivo. These findings provide molecular insights into the functional divergence of CYP76 family enzymes and establish a foundation for metabolic engineering of valuable MIAs.