Functional characterization of Camptotheca acuminata 7-deoxyloganetic acid synthases and 7-deoxyloganetic acid glucosyltransferases involved in camptothecin biosynthesis
Camptothecin (CAM), a well-known plant-derived antitumor compound, is a structurally complex pentacyclic pyrroloquinoline monoterpene indole alkaloid (MIA) found in various plant species. As a specific MIA, CAM had been thought to share a common upstream biosynthetic pathway with other MIAs such as...
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Veröffentlicht in: | Plant physiology and biochemistry 2025-01, Vol.218, p.109305, Article 109305 |
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Zusammenfassung: | Camptothecin (CAM), a well-known plant-derived antitumor compound, is a structurally complex pentacyclic pyrroloquinoline monoterpene indole alkaloid (MIA) found in various plant species. As a specific MIA, CAM had been thought to share a common upstream biosynthetic pathway with other MIAs such as the antitumor vinblastine and vincristine from Catharanthus roseus. Nevertheless the key enzymes responsible for the consecutive three-step oxidation of the –CH3 of nepetalactol to form the –COOH of 7-deoxyloganetic acid and the subsequent glycosylation of 7-deoxyloganetic acid to yield 7-deoxyloganic acid have yet to be functionally characterized. Here we established an in vivo tandem catalysis assay for the enzymatic catalytic activity characterization of 7-deoxyloganetic acid synthase (7DLS) and 7-deoxyloganetic acid glucosyltransferase (7DLGT), two crucial catalytic enzymes in MIAs biosynthesis, thereby avoiding the difficulty in the detection of the unstable biosynthetic intermediates. The enzyme activity assay platform was conducted through the co-expression of functionally characterized Cr7DLS and Cr7DLGT in Saccharomyces cerevisiae WAT11, substrate feeding, and enzymatic product verification. Two cytochrome P450 enzymes (CYPs) from Camptotheca acuminata, the prestigious resource for CAM, CaCYP76A75 and CaCYP76A76, were identified and functionally characterized to be responsible for the consecutive three-step oxidation of nepetalactol to yield 7-deoxyloganetic acid through reciprocal replacement of Cr7DLS in the in vivo tandem enzyme activity assay platform. Two uridine 5′-diphosphate glycosyltransferases (UGTs), CaUGT709C10 and CaUGT709C11, were functionally characterized to be capable of glycosylating 7-deoxyloganetic acid to yield 7-deoxyloganic acid. This study provides two CYPs as 7DLSs and two UGTs as 7DLGTs, offering potential applications in MIAs biosynthesis.
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•An in vivo tandem catalysis was established for enzyme's functional characterization.•CYP76A75 and CYP76A76 were identified and functionally characterized as Ca7DLSs.•UGT709C10 and UGT709C11 were identified and functionally characterized as Ca7DLGTs. |
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ISSN: | 0981-9428 1873-2690 1873-2690 |
DOI: | 10.1016/j.plaphy.2024.109305 |