Three cytochrome P450 88A subfamily enzymes, CYP88A108, CYP88A164, and CYP88A222, act as β-amyrin 11-oxidases involved in triterpenoid biosynthesis in Melia azedarach L.

Abstract

Melia azedarach L. serves as an important source of timber and bioactive triterpenoids, notably tetracyclic limonoids and pharmacologically active pentacyclic triterpenes such as β-amyrin derivatives. Although limonoid biosynthesis has been extensively characterized, the enzymatic machinery governing pentacyclic triterpene formation remains poorly defined. Through transcriptomic mining of M. azedarach, we identified three CYP88A subfamily cytochrome P450 genes (MaCYP88A108, MaCYP88A164, and MaCYP88A222). Functional characterization in both Nicotiana benthamiana transient expression systems and yeast microsomes demonstrated their catalytic roles as β-amyrin 11-oxidases, sequentially converting β-amyrin to 11-oxo-β-amyrin via C-11 oxidation. Intriguingly, MaCYP88A108 and MaCYP88A164 exhibited dual functionality, participating not only in pentacyclic triterpene production but also in limonoid biosynthesis, suggesting their evolutionary recruitment for triterpenoid diversification. Phylogenetic reconstruction and structural modeling revealed that these CYP88A enzymes share close homology and conserved active-site architectures with Glycyrrhiza uralensis CYP88D6 (GuCYP88D6), a characterized β-amyrin 11-oxidase, despite lacking genomic synteny. This observation supports a paradigm of functional convergence driven by structural conservation rather than shared gene lineage. Furthermore, we characterized an oxidosqualene cyclase (MaβAS1) responsible for cyclizing 2,3-oxidosqualene to β-amyrin, thereby establishing the substrate pool for downstream oxidation. Our study uncovers a unique catalytic versatility in plant CYP88A P450s, demonstrating their capacity to coordinate two divergent triterpenoid branches. This functional plasticity, mediated by evolutionarily conserved structural frameworks, expands current paradigms of enzyme multifunctionality in plant specialized metabolism. The identified genes (CYP88As and MaβAS1) establish critical tools for synthetic biology platforms targeting heterologous production of pharmaceutically important triterpenoids, bridging mechanistic exploration of plant secondary metabolism with biotechnological applications.