Discovery of unique CYP716C oxidase involved in pentacyclic triterpene biosynthesis from Camptotheca acuminata.

Dozens of triterpenes have been isolated from Camptotheca acuminata, however, triterpene metabolism in this plant remains poorly understood. The common C28 carboxy located in the oleanane-type and ursane-type triterpenes indicates the existence of a functionally active triterpene, C28 oxidase, in this plant. Thorough mining and screening of the CYP716 genes were initiated using the multi-omics database for C. acuminata. Two CYP716A (CYP716A394 and CYP716A395) and three CYP716C (CYP716C80-CYP716C82) were identified based on conserved domain analyses and hierarchical cluster analyses. CYP716 microsomal proteins were prepared and their enzymatic activities were evaluated in vitro. The CYP716 classified into the CYP716C subfamily displays ß-amyrin oxidation activity, and CYP716A displays α-amyrin and lupeol oxidation activity, based on gas chromatography-mass spectrometry analyses. The oxidation products were determined based on their mass and nuclear magnetic resonance spectrums. The optimum reaction conditions and kinetic parameters for CYP716C were determined, and functions were verified in Nicotiana benthaminana. Relative quantitative analyses revealed that these CYP716C genes were enriched in the leaves of C. acuminata plantlets after 60 d. These results indicate that CYP716C plays a dominant role in oleanane-type triterpene metabolism in the leaves of C. acuminata via a substrate-specific manner, and CYP716A is responsible for ursane- and lupane-type triterpene metabolism in fruit. This study provides valuable insights into the unique CYP716C-mediated oxidation step of pentacyclic triterpene biosynthesis in C. acuminata.

Proteomics-Guided Mining and Characterization of Epoxidase Involved in Camptothecin Biosynthesis from Camptotheca acuminata.

The detailed metabolic map for camptothecin (CPT) biosynthesis in Camptotheca acuminata has been proposed according to our combined omics results. However, the CYP450-mediated epoxidation step in CPT biosynthesis remains unexplored. A proteomics-guided approach was used to identify and annotate the proteins enriched during the vigorous CPT metabolism period in mature C. acuminata and seedlings. Comparative analyses revealed that the CPT and flavonoid biosyntheses were vigorous in stems and all of the samples except the leaves, respectively. The CYP71BE genes were screened based on their enrichment patterns at the transcriptomic-proteomic level and biochemically characterized in Saccharomyces cerevisiae WAT11. Four CYP71BE proteins exhibited in vitro isoliquiritigenin epoxidase activity. Additionally, CYP71BE206 showed epoxidase activity toward strictosamide, the critical precursor for CPT biosynthesis, both in vitro and in Nicotiana benthamiana. In planta functional verification suggested that CYP71BE206 is involved in CPT biosynthesis. Their catalytic conditions were optimized, and the enzymatic parameters were determined. This study provides valuable insight into the CYP71BE-mediated epoxidation step for CPT biosynthesis and offers evidence to verify that the newly characterized epoxidase (CYP71BE206) is simultaneously responsible for the biosynthesis of CPT and the flavonoid in this plant. An evolution event probably happened on ancestral CYP71BE, resulting in the neofunctionalization of CYP71BE206.

Synthesis and anti-tumor activity study of water-soluble PEG-celastrol coupling derivatives as self-assembled nanoparticles.

To improve the drug-ability of celastrol, a series of PEGylation celastrol (PEGC) were designed and synthesized by conjugation with different kinds of polyethylene glycols (PEGs) with celastrol. Most of PEGCs could easily dissolve in water. In particular, one of them (DC1000) could be dispersed in water to form nanoparticles by self-assembly. The cytotoxic evaluation of PEGCs revealed that some of PEGCs showed more potent cytotoxicity than celastrol, and the molecular weight of PEG parts in PEGCs had apparent influence on their cytotoxic activity. Anti-tumor evaluation in vivo showed DC1000 had higher tumor inhibition rate and better safety than celastrol by intravenous administration with equivalent molar weight. These results revealed PEGylation might be an efficient and economical method to improve the water solubility and safety of celastrol and similar natural products.

Synthesis of 3- and 29-substituted celastrol derivatives and structure-activity relationship studies of their cytotoxic activities.

A series of 3-carbamate and 29-ester celastrol derivatives (compounds 1-26) were designed and synthesized. These analogues were evaluated for their cytotoxic activities against several cancer cell lines. Cytotoxicity data revealed that the properties of substituents and substitution position had important influence on cytotoxic activity. Modification of C-3 hydroxyl with size-limited groups did not reduce the activity obviously. The introduction of polarity group like piperazine could improve the solubility. Compound 23 was chosen to further evaluate anti-tumor efficacy in vivo. It showed higher inhibition rate and better safety than celastrol during in vivo experiment by intragastric administration. The preliminary antitumor studies of compound 23in vivo showed that it might be promising for the development of new antitumor agents.