Characterization of oxidosqualene cyclases from Trichosanthes cucumerina L. reveals key amino acids responsible for substrate specificity of isomultiflorenol synthase.

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CONCLUSION: Two key amino acids of isomultiflorenol synthase, Y125 and M254, were first proposed. They could be associated with the production of isomultiflorenol. Oxidosqualene cyclases (OSCs) are the first committed enzymes in the triterpenoid biosynthesis by converting 2,3-oxidosqualene to specific triterpenoid backbones. Thus, these enzymes are potential targets for developing plant-active compounds through the study of triterpenoid biosynthesis. We applied transcriptome information and metabolite profiling from Trichosanthes cucumerina L. to define the diversity of triterpenoids in this plant through OSCs. Isomultiflorenol synthase and cucurbitadienol synthase were previously identified in this plant. Here, three new OSCs, TcBAS, TcLAS, and TcCAS, were cloned and functionally characterized as β-amyrin synthase, lanosterol synthase, and cycloartenol synthase activities, respectively. We also took advantage of the multiple sequence alignment and molecular docking of OSCs exhibiting in this plant and other plant OSCs to identify key residues associated with isomultiflorenol synthase specificity. Two novel key amino acids, referred to the Y125 and M254, were first discovered. These results provide information on a possible catalytic mechanism for plant OSCs that produce specific products.