Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa

ABSTRACT Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity. Given that two C‐10 modified camptothecin derivatives, topotecan and irinotecan, have been approved as potent anticancer agents, there is a critical need for methods to access other aromatic ring‐functionalized congeners (e.g., C‐9, C‐10, etc.). However, contemporary methods for chemical oxidation are generally harsh and low‐yielding when applied to the camptothecin scaffold, thereby limiting the development of modified derivatives. Reported herein, we have identified four tailoring enzymes responsible for C‐9 modifications of camptothecin from Nothapodytes tomentosa , via metabolomic and transcriptomic analysis. These consist of a cytochrome P450 ( Nt CPT9H) which catalyzes the regioselective oxidation of camptothecin to 9‐hydroxycamptothecin, as well as two methyltransferases ( Nt OMT1/2, converting 9‐hydroxycamptothecin to 9‐methoxycamptothecin), and a uridine diphosphate‐glycosyltransferase ( Nt UGT5, decorating 9‐hydroxycamptothecin to 9‐ β ‐D‐glucosyloxycamptothecin). Importantly, the critical residues that contribute to the specific catalytic activity of Nt CPT9H have been elucidated through molecular docking and mutagenesis experiments. This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering. This will hasten the discovery of novel C‐9 modified camptothecin derivatives, with profound implications for pharmaceutical manufacture.