Functional characterization of a glycoside hydrolase in the biosynthesis of camptothecin from Camptotheca acuminata

Camptothecin, a plant-derived pentacyclic pyrroloquinoline alkaloid, and its derivatives like topotecan and irinotecan have been used as clinical anticancer agents for decades. However, the complete biosynthetic pathway of camptothecin still remains unelucidated due to the unknown complex formation processes and corresponding enzymes for the downstream biosynthetic pathway including the committed hydrolysis of glycosides. Herein, a novel glycoside hydrolase (CaGH1) responsible for the deglycosylation of biosynthetic glycoside intermediates including both quinoline-type alkaloids pumiloside (1), (3S)-deoxypumiloside (2) and indole-type alkaloid strictosamide (3) has been functionally identified. Moreover, CaGH1 exhibits the highly strict stereoselectivity towards the substrates with 3S configuration. Furthermore, a combined strategy for the discovery of the unknown biosynthetic enzyme by employing activity-guided enzyme verification, transcriptome-based gene mining, biochemical assay in vitro, and structurally characterizing the unstable enzymatic products by derivatization, is reported. These findings not only provide a better understanding of the deglycosylation in camptothecin biosynthesis, also lay the foundation for the complete elucidation of camptothecin biosynthetic pathway and biological production of camptothecin. A glycoside hydrolase with stereoselective catalytic activity towards substrates with 3S configuration including quinoline-type alkaloids (pumiloside and (3S)-deoxypumiloside, 1 and 2) and indole-type alkaloid (strictosamide, 3) in the biosynthesis of camptothecin has been identified from Camptotheca acuminata. [Display omitted]