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 o...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.展开更多
基金financially supported by the National Key Research and Development Program of China(No.2020YFA0908000)CAMS Innovation Fund for Medical Sciences(No.CIFMS-2023I2M-2–006)。
文摘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.
