Chemoproteomics reveals the epoxidase enzyme for the biosynthesis of camptothecin in <i>Ophiorrhiza pumila</i>

Abstract

Camptothecin is one of the most commonly used anticancer drugs worldwide, yet the downstream biosynthetic route from strictosidine to camptothecin has remained unclear for more than half a century. Here, we searched for proteins involved in camptothecin biosynthesis from the camptothecin-producing plant Ophiorrhiza pumila by chemoproteomics and identified OpCYP716E111. Exogenously expressing OpCYP716E111 in Nicotiana benthamiana and yeast (Saccharomyces cerevisiae) led to the production of strictosamide epoxide 2 from strictosamide 1. Our findings thus reveal the enzyme responsible for the biosynthesis of strictosamide epoxide in the camptothecin biosynthetic pathway. In addition, our results highlight the potential of using chemoproteomics as a tool for discovering enzymes involved in natural product biosynthesis. Camptothecin and its derivatives comprise the third largest class of anticancer drugs in the worldwide market. These drugs are used to treat malignant tumors such as lung, colon, and cervical cancer. Camptothecin was first discovered in Camptotheca acuminata and is commonly extracted mainly from C. acuminata and Nothapodytes nimmoniana for medicinal use (Sadre et al., 2016). However, the overharvesting of C. acuminata and N. nimmoniana has greatly reduced their populations in nature, and they are currently listed as endangered plants in China and India. It is estimated that 20 million new cancer cases will appear in 2025 worldwide. Therefore, meeting the growing demand for camptothecin and other anticancer drugs has become a daunting challenge (Seca and Pinto, 2018). Due to the limited availability of natural plant resources and the high cost of chemical synthesis, producing camptothecin using synthetic biology based on the elucidation of biosynthetic pathways has become an appealing strategy for achieving this goal. However, the biosynthetic pathway of camptothecin has not yet been completely elucidated. It is thought that strictosidine is first converted to strictosamide via a reduction reaction and that strictosamide 1 is oxidized to strictosamide epoxide 2 (Kang et al., 2021). Strictosamide epoxide 2 would then be converted to camptothecin via a series of oxidation, reduction, and other reactions (Figure S1). A combination of genomic, transcriptomic, and proteomic analyses can be utilized to screen candidate genes and to elucidate the unknown biosynthetic pathways of natural plant products (He et al., 2023). These omics data-based strategies have been successful in many cases. However, such methods often generate many candidates, making it quite laborious and costly to screen target genes. Chemoproteomic profiling, a method based on investigating protein–small molecule interactions by combining functionalized chemical probes and proteomic analysis, has emerged as a promising tool to mine target enzymes in the biosynthetic pathways of natural products. Here, using this approach, we discovered the enzyme responsible for the biosynthesis of strictosamide epoxide 2 in the camptothecin biosynthetic pathway. Our findings lay the foundation for synthetic biological synthesis of this important drug and highlight the effectiveness of chemoproteomic profiling for discovering unknown enzymes involved in the biosynthesis of natural products. In this study, we aimed to elucidate the unknown biosynthetic pathway from strictosamide 1 to strictosamide epoxide 2 by unearthing the responsible enzymes based on the sequences of O. pumila proteins using the chemoproteomic strategy (Figure 1A). We designed a bait known as ST-probe 4, with a diazirine-alkyne tag, and synthesized it via 1,3-dicyclohexylcarbodiimide to promote the condensation of strictosamide 1 and 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetic acid (Dead-probe 3) in anhydrous dimethylformamide (DMF) (Figures S3, S4). The structure of the synthetic probe was confirmed by HR-MS and NMR (Figures S5–10). Identification of a CYP450 enzyme that catalyzes the formation of strictosamide epoxide 2 from strictosamide 1 by chemoproteomic analysis (A) Image of a 6-month-old Ophiorrhiza pumila plant. (B) Synthesis of probe ST-probe 4. (C) Workflow of chemoproteomic profiling of O. pumila extract using ST-probe 4. (D) Effect of enrichment using ST-probe 4/Dead-probe 3. Red dots represent proteins with increased accumulation, green dots represent proteins with decreased accumulation, gray dots represent proteins showing no change in levels using ST-probe 4 versus Dead-probe 3. (E) LC/MS analysis of the newly produced compound in extracts from N. benthamiana leaves transiently expressing OpCYP716E111 versus empty vector (EV); extracted ion chromatograms for strictosamide epoxide 2 (m/z [M + H]+ = 515.2024) are shown. (F) MS/MS spectra of strictosamide epoxide 2 produced in N. benthamiana. (G) The reaction catalyzed by OpCYP716E111. (H) LC/MS analysis of the newly produced compound in Saccharomyces cerevisiae expressing OpCYP716E111; extracted ion chromatograms for strictosamide epoxide 2 (m/z [M + H]+ = 515.2024) are shown. (I) Phylogenetic analysis of OpCYP716E111 and other P450 enzymes. To identify the corresponding protein that catalyzes the production of strictosamide epoxide 2 from strictosamide 1, we harvested 6-month-old O. pumila plants and extracted crude proteins (Figure 1B). We incubated the crude protein extract with 100 μM ST-probe 4 or Dead-probe 3 (a negative control probe without the strictosamide motif; Gao et al., 2020). We then subjected the proteins to biotinylation, enriched and digested the proteins, and identified and quantified the peptide fragments by LC–MS/MS (Zhou et al., 2018). We identified 296 proteins from the incubated O. pumila proteins with a probe (Figure 1C). Five proteins demonstrated a more than two-fold change in level (considered to be significant hits) in the ST-probe group in a volcano plot (Table S1), including a protein named OpCYP716E111, which was annotated as a CYP450 enzyme (Figure 1D). We detected the peptide fragment LLTSWWPQSMK, which was only a partial sequence of OpCYP716E111 predicted from transcriptomic data of O. pumila (Table S2). The four other proteins were annotated as a thaumatin-like protein, ATP synthase, superoxide dismutase, and hypothetical protein, which we considered less likely to catalyze the epoxidation reactions. Therefore, we selected OpCYP716E111 for further analysis as a candidate enzyme to produce strictosamide epoxide 2. To investigate the biochemical activity of OpCYP716E111, we cloned its corresponding gene (OpCYP716E111) from cDNA isolated from an O. pumila plant and transformed N. benthamiana leaves with a construct harboring this gene for transient expression. After feeding the N. benthamiana with strictosamide for 72 h, we subjected the leaves to methanol extraction. A chromatographic peak with a molecular weight of (M + H)+ = 515.2024, the same molecular weight as the target compound strictosamide epoxide 2, was detected in extracts from N. benthamiana leaves expressing OpCYP716E111, but not from leaves transformed with empty vector (Figures 1E, S11). The major ionic fragments of the newly produced compound—515, 353, 335, 283, 265, and 237—were the same as those previously reported for strictosamide epoxide 2 (Sadre et al., 2016; Figure S2), supporting the conclusion that OpCYP716E111 catalyzes the production of strictosamide epoxide 2 from strictosamide 1 (Figure 1F–G). To further explore the function of OpCYP716E111, we exogenously overexpressed OpCYP716E111 and Arabidopsis thaliana ATR2 (encoding cytochrome P450 reductase) in yeast (Saccharomyces cerevisiae) driven by separately inducible promoters. The crude enzyme and microsomes from yeast expressing OpCYP716E111 catalyzed the production of strictosamide epoxide 2 from strictosamide 1 (Figure 1H). Phylogenetic analysis revealed that OpCYP716E111 belongs to the CYP716 family (Figure 1I). The CYP716 family cytochrome P450 reductase PgCYP716S5 from Platycodon grandiflorus was previously shown to catalyze the epoxidation of β-amyrin into 12,13α-epoxy β-amyrin (Miettinen et al., 2021). A recent study demonstrated that the CYP71 family gene CYP71BE206 from C. acuminata had strictosamide epoxidase activity when transiently expressed in N. benthamiana (Pu et al., 2023). OpCYP716E111 and CYP71BE206 are not homologous genes as they share only 20.7% of the sequence identity, expanding our understanding of the catalysis process of plant secondary metabolism. In the camptothecin-producing plant O. pumila, the indole synthetic pathway providing the precursor tryptamine and the iridoid pathway providing the precursor secologanin have been elucidated (Hao et al., 2023). The formation of strictosidine from tryptamine and secologanin is catalyzed by strictosidine synthetase, however the downstream biosynthetic route from strictosidine to camptothecin has remained unclear for more than half a century (Rai et al., 2021). O. pumila contains ~24,000 genes based on the annotation of its transcriptome. We successfully reduced the number of candidate proteins to 296 based on chemoproteomics profiling analysis. Our further results confirmed OpCYP716E111 as the enzyme responsible for producing strictosamide epoxide 2. Notably, this work showcases chemoproteomics as an efficient tool for discovering unknown enzymes involved in natural product biosynthesis. This work was supported financially by the National Key Research and Development Program of China (Grant No. 2018YFA0900700), the National Natural Science Foundation of China (Nos. 31901021; 31921006), and the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences (Grant No. KFJ-BRP-009). We thank Xiaoyan Xu and Shanshan Wang in the Core Facility Centre of CEMPS for their help in analyzing chemical structures and protein sequences. We thank Dr. David Nelson from the University of Tennessee for naming the gene OpCYP716E111. The authors declare no conflict of interest. P.W., X.Y., Z.Z., and T.Z. designed the research. T.Z. performed the experiments, generated the figure, analyzed the data, and wrote the manuscript under the guidance of P.W., Z.Z. and Y.W. designed and synthesized the chemical probe. S.W. and C.Y. performed the protein expression and functional characterization. E.T. extracted the crude enzymes from plants. All authors contributed a lot to this work and approved the final version of the manuscript. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13594/suppinfo Figure S1. The proposed biosynthetic pathway of camptothecin in Ophiorrhiza pumila Figure S2. MS spectra and cleavage patterns of epoxidation products for OpCYP716E111 Figure S3. The 1H NMR spectrum for strictosamide 1 Figure S4. The 1H NMR spectrum for Dead-probe 3 Figure S5. Synthesis of probe ST-probe 4 Figure S6. 1H NMR spectrum of the probe ST-probe 4 Figure S7. 13C NMR spectrum of the probe ST-probe 4 Figure S8. HMBC spectrum of the probe ST-probe 4 Figure S9. COSY spectrum of the probe ST-probe 4 Figure S10. HSQC spectrum of the probe ST-probe 4 Figure S11. Analysis of new product Table S1. Five proteins with a fold change higher than 2 Table S2. Some homology sequences to OpCYP716E111 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Topics & Concepts