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Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle

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Science  15 Jun 2018:
Vol. 360, Issue 6394, pp. 1235-1239
DOI: 10.1126/science.aat4100

How to make bioactive alkaloids

Vinblastine and vincristine are important, expensive anticancer agents that are produced by dimerization of the plant-derived alkaloids catharanthine and vindoline. The enzymes that transform tabersonine into vindoline are known; however, the mechanism by which the scaffolds of catharanthine and tabersonine are generated has been a mystery. Caputi et al. now describe the biosynthetic genes and corresponding enzymes responsible. This resolves a long-standing question of how plant alkaloid scaffolds are synthesized, which is important not only for vinblastine and vincristine biosynthesis, but also for understanding the many other biologically active alkaloids found throughout nature.

Science, this issue p. 1235

Abstract

Vinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.

Anticancer drugs vincristine 5 and vinblastine 6 were discovered 60 years ago in Catharanthus roseus (Madagascar periwinkle). These compounds have been used for the treatment of several types of cancer, including Hodgkin’s lymphoma, as well as lung and brain cancers. Much of the metabolic pathway (31 steps from geranyl pyrophosphate to vinblastine) has been elucidated (13). Here we report the genes encoding the missing enzymes that complete the vinblastine pathway. Two redox enzymes convert stemmadenine acetate 7 into an unstable molecule, likely dihydroprecondylocarpine acetate 11, which is then desacetoxylated by one of two hydrolases to generate, through Diels-Alder cyclizations, either tabersonine 2 or catharanthine 3 scaffolds that are ultimately dimerized to yield vinblastine and vincristine (Fig. 1A). In addition to serving as the precursors for vincristine 5 and vinblastine 6, tabersonine 2 and catharanthine 3 are also precursors for other biologically active alkaloids (4, 5) (Fig. 1B). The discovery of these two redox enzymes (precondylocarpine acetate synthase and dihydroprecondylocarpine acetate synthase), along with the characterization of two hydrolases (tabersonine and catharanthine synthase), provides insight into the mechanisms that plants use to create chemical diversity and also enables production of a variety of high-value alkaloids.

Fig. 1 Vincristine and vinblastine biosynthesis.

(A) Vincristine 5 and vinblastine 6 are formed by dimerization from the monomers catharanthine 3 and vindoline 4 by a peroxidase (28) or chemical methods (29). The genes that convert tabersonine 2 to vindoline 4 have been identified (24). (B) Representative bioactive alkaloids derived from stemmadenine. Me, methyl; Et, ethyl.

Catharanthine 3 (iboga-type alkaloid) and tabersonine 2 (aspidosperma-type) scaffolds are likely generated by dehydration of the biosynthetic intermediate stemmadenine 1 to dehydrosecodine 9, which can then cyclize to either catharanthine 3 or tabersonine 2 via a net [4+2] cycloaddition reaction (Fig. 2) (69). We speculated that the missing components were an enzyme with dehydration and cyclization function. We hypothesized that the unstable nature of the dehydration product dehydrosecodine 9 (7) would preclude its diffusion out of the enzyme active site, and we thus searched for an enzyme that could catalyze both dehydration and cyclization reactions.

Fig. 2 Biosynthesis of catharanthine and tabersonine scaffolds.

Stemmadenine acetate 7 {generated from stemmadenine 1 under condition i [Ac2O (excess) and pyridine (excess) at room temperature (r.t.), 4 hours, >99% yield]} undergoes an oxidation to form precondylocarpine acetate 10. This reaction is catalyzed enzymatically by the reticuline oxidase homolog PAS or, alternatively, can be generated synthetically under condition ii [Pt (from 7.5 equivalents of PtO2), EtOAc, O2 atmosphere, r.t., 10 hours, yields varied], as reported by Scott and Wei (8). Next, the open form of precondylocarpine acetate 10 is reduced by the alcohol dehydrogenase DPAS. This reduced intermediate could not be isolated due to its lability but, on the basis of degradation product tubotaiwine 12, is assumed to be dihydroprecondylocarpine acetate 11. Dihydroprecondylocarpine acetate 11, in the open form, can form dehydrosecodine 9 through the action of CS or TS to form catharanthine 3 or tabersonine 2, respectively. Numbers within structures indicate different carbon atoms. spont., spontaneous; HRMS, high-resolution mass spectrometry; MS/MS, tandem mass spectrometry.

Because the biosynthetic genes for vincristine 5 and vinblastine 6 are not clustered in the plant genome (10), we searched for gene candidates in RNA sequencing (RNA-seq) data (10) from the vincristine- and vinblastine-producing plant C. roseus. Two genes annotated as alpha/beta hydrolases were identified by a shared expression profile with previously identified vinblastine biosynthetic enzymes (fig. S1A and data S1). A dehydratase could facilitate the isomerization of the 19,20-exo-cyclic double bond of stemmadenine 1 to form iso-stemmadenine 8 (Fig. 2), which would then allow dehydration to form dehydrosecodine 9 and, consequently, catharanthine and tabersonine (8). Virus-induced gene silencing (VIGS) of herein-named tabersonine synthase (TS) and catharanthine synthase (CS) (Fig. 1 and figs. S2 and S3) in C. roseus resulted in a marked reduction of tabersonine 2 (P= 0.0048) and catharanthine (P = 0.01), respectively. These silencing experiments implicate CS and TS in catharanthine 3 and tabersonine 2 biosynthesis in C. roseus. However, when CS and TS were heterologously expressed in Escherichia coli (fig. S4A) and tested for reactivity with stemmadenine 1 (fig. S5) or the acetylated form of stemmadenine 7 (fig. S6), in which spontaneous deformylation would be hindered (Fig. 2, figs. S5 and S6, and tables S4 and S5), no reaction was observed. Although TS and CS are known to be implicated in vinblastine biosynthesis (11), the substrates, and therefore the specific catalytic functions, remained elusive.

We used enzyme-assay guided fractionation in an attempt to isolate the active substrate for the TS and CS enzymes from various aspidosperma-alkaloid– and iboga-alkaloid–producing plants. We focused on Tabernaemontana plants that are known to accumulate more stemmadenine 1 intermediate relative to downstream alkaloids (12). These experiments demonstrated that TS and CS were always active with the same fractions (fig. S7), consistent with previous hypotheses (6) that both enzymes use the same substrate. However, attempts to structurally characterize the substrate were complicated by its rapid decomposition, and the deformylated product tubotaiwine 12 [previously synthesized in reference (13)] was the major compound detected in the isolated mixture [Fig. 2 and nuclear magnetic resonance (NMR) data in fig. S8]. Given the propensity for deformylation in these structural systems (14), we rationalized that tubotaiwine 12 could be the decomposition product of the actual substrate, which would correspond to iso-stemmadenine 8 (dihydroprecondylocarpine) or its protected form (dihydroprecondylocarpine acetate 11) (Fig. 2). We surmised that a coupled oxidation-reduction cascade could perform a net isomerization to generate dihydroprecondylocarpine 8 (or dihydroprecondylocarpine acetate 11) from stemmadenine 1 (or stemmadenine acetate 7). This idea was initially proposed by Scott and Wei, who indicated that stemmadenine acetate 7 can be oxidized to precondylocarpine acetate 10, after which the 19,20-double bond can then be reduced to form dihydroprecondylocarpine acetate 11, which can then form traces of tabersonine 2 upon thermolysis (15). Similar reactions with stemmadenine 1 resulted in deformylation to form condylocarpine 13 (16). Therefore, we reexamined the RNA-seq dataset for two redox enzymes that could convert stemmadenine acetate 7 to dihydroprecondylocarpine acetate 11.

We noted a gene annotated as reticuline oxidase that had low absolute expression levels but a similar tissue expression pattern to that of the TS gene (fig. S1B). The chemistry of reticuline oxidase enzymes (17) such as berberine bridge enzyme and dihydrobenzophenanthridine oxidase suggests that these enzymes are capable of C–N bond oxidation, which would be required in this reaction sequence (Fig. 2) (17). When this oxidase gene was silenced in C. roseus, a compound with a mass and 1H NMR spectrum corresponding to semisynthetically prepared stemmadenine acetate 7 (the proposed oxidase substrate) accumulated, suggesting that this gene encoded the correct oxidase. We named this enzyme precondylocarpine acetate synthase (PAS) (figs. S9 to S11). Similarly, silencing of a medium-chain alcohol dehydrogenase, as part of an ongoing screen of alcohol dehydrogenases in C. roseus (14, 18, 19), resulted in accumulation of a compound with a mass, retention time, and fragmentation pattern consistent with a partially characterized synthetic standard of precondylocarpine acetate 10 (the proposed substrate of the reductase) (figs. S12 to S14). This standard could be synthesized from stemmadenine acetate 7 using Pt and O2 by established methods (8, 15, 20). With our small-scale reactions, yields were low and variable, and the product decomposed during characterization. However, the limited two-dimensional NMR dataset was consistent with an assignment of precondylocarpine acetate 10. Thus, we renamed this alcohol dehydrogenase dihydroprecondylocarpine synthase (DPAS). Collectively, these data suggest that PAS and DPAS act in concert with CS or TS to generate catharanthine 3 and tabersonine 2.

To validate whether these enzymes produce catharanthine 3 and tabersonine 2, we transiently coexpressed PAS, DPAS, and CS or TS in the presence of stemmadenine acetate 7 in Nicotiana benthamiana. These experiments illustrated the sequential activity of the newly discovered enzymes, whereby we observed formation of catharanthine 3 in plant tissue overexpressing PAS, DPAS, and CS or tabersonine 2 in plant tissue overexpressing PAS, DPAS, and TS, when the leaf was also coinfiltrated with stemmadenine acetate 7 (Fig. 3A). The presence of all proteins was validated by proteomics analysis (fig. S4B and data S2). Formation of precondylocarpine acetate 10 was observed when stemmadenine acetate 7 was infiltrated into N. benthamiana in the absence of any heterologous enzymes (Fig. 3A), suggesting that an endogenous redox enzyme(s) of N. benthamiana can oxidize stemmadenine acetate 7. Formation of 3 and 2 was validated by coelution with commercial standards, and formation of 10 was validated by coelution with the semisynthetic compound.

Fig. 3 Biosynthesis of tabersonine 2 and catharanthine 3 from stemmadenine acetate 7 starting substrate.

(A) Reconstitution of tabersonine 2 and catharanthine 3 in N. benthamiana from stemmadenine acetate 7. Extracted ion chromatograms (XIC) for ions with mass/charge ratio (m/z) 397.19 (stemmadenine acetate 7), m/z 395.19 (precondylocarpine acetate 10), and m/z 337.19 [catharanthine at retention time (RT) 4.0 min and tabersonine at RT 4.4 min] are shown. EV, empty vector. (B) Interaction of CS and TS with DPAS by bimolecular fluorescence complementation (BiFC) in C. roseus cells. The efficiency of BiFC complex reformation reflected by the YFP fluorescence intensity highlighted that CS and DPAS exhibited weak interactions (i to iii), whereas TS and DPAS strongly interacted (iv to vi). No interactions with loganic acid methyltransferase (LAMT) were observed (vii to ix). YN, YFP N-terminal fragment; YC, YFP C-terminal fragment; nuc-CFP, nuclear cyan fluorescent protein; DIC, differential interference contrast. Scale bars, 10 μm. (C) Phylogenetic relationship of PAS with other functionally characterized berberine bridge enzymes.

Purified proteins were required to validate the biochemical steps of this reaction sequence in vitro. Whereas CS, TS, and DPAS all expressed in soluble form in E. coli (fig. S4A), the flavin-dependent enzyme PAS failed to express in standard expression hosts such as E. coli or Saccharomyces cerevisiae. To overcome this obstacle, we expressed the native full-length PAS in N. benthamiana plants using a transient expression system (fig. S4B) in Pichia pastoris (fig. S4, C and D) and in Sf9 insect cells (fig. S4E). The presence of PAS was validated by proteomic data (data S3). Reaction of PAS from each of these expression hosts with stemmadenine acetate 7 produced a compound that had an identical mass and retention time to our semisynthetic standard of precondylocarpine acetate 10 (figs. S15 and S16). The enzymatic assays with PAS protein derived from P. pastoris and Sf9 insect cells ensure that formation of the expected products is not the result of a protein contaminant found in the plant-expressed PAS protein. When the PAS proteins (from N. benthamiana and P. pastoris), along with stemmadenine acetate 7, were combined with heterologous DPAS and CS, catharanthine 3 was formed, and when combined with DPAS and TS, tabersonine 2 was observed (figs. S17 and S18).

Semisynthetic precondylocarpine acetate 10 could be reacted with DPAS and TS or CS to yield tabersonine 2 or catharanthine 3, respectively (fig. S19). In addition, a crude preparation of what is proposed to be dihydroprecondylocarpine acetate 11, synthesized according to Scott and Wei (8), was converted to catharanthine 3 and tabersonine 2 by the action of CS and TS, respectively (fig. S20). Reaction of PAS (purified from N. benthamiana) and DPAS with stemmadenine acetate 7 in the absence of CS or TS yielded a compound isomeric to tabersonine 2 and catharanthine 3, suggesting that cyclization can occur spontaneously under these reaction conditions (fig. S17). As observed during attempts to purify the CS or TS substrate from Tabernaemontana plants, dihydroprecondylocarpine acetate 11 can also deformylate to form tubotaiwine 12. Solvent and reaction conditions probably determine how the reactive dihydroprecondylocarpine acetate 11 decomposes.

PAS failed to react with stemmadenine 1, indicating that the enzyme recognized the acetyl (Ac) group (fig. S21). Oxidation of stemmadenine 1 produced a compound with a mass consistent with that of the shunt product condylocarpine 13. This identification was supported by comparison of the tandem mass spectrometry spectrum of 13 to that of the related compound tubotaiwine 12 (fig. S22). The transformation of stemmadenine 1 to condylocarpine 13 is known (15, 21). We therefore suspect that the acetylation of stemmadenine 1 is necessary to slow spontaneous deformylation after oxidation, and AcOH serves as a leaving group to allow formation of dehydrosecodine 9 (20). Acetylation also functions as a protecting group in the biosynthesis of noscapine in opium poppy (22).

The reactivity of the intermediates involved in the transformation of stemmadenine acetate 7 to catharanthine 3 or tabersonine 2 suggests that PAS, DPAS, and CS or TS should be colocalized because the unstable post–precondylocarpine acetate 10 intermediates may not remain intact during transport between cell types or compartments. Using yellow fluorescent protein (YFP)–tagged proteins in C. roseus cell suspension culture, we showed that PAS is targeted to the vacuole through small vesicles budding from the endoplasmic reticulum (ER), as was previously observed for the PAS homolog, berberine bridge enzyme (23) (fig. S23). This localization suggests that stemmadenine acetate 7 oxidation occurs in the ER lumen, ER-derived vacuole-targeted vesicles, and/or vacuole. In contrast, colocalization of DPAS, CS, and TS was confirmed in the cytosol (figs. S24 and S25). Bimolecular fluorescence complementation suggested preferential interactions between DPAS and TS (Fig. 3B and figs. S26 and S27). Such interactions may not only prevent undesired reactions on the reactive dihydroprecondyocarpine acetate 11 intermediate but may also control the flux of 11 into tabersonine 2.

Homologs of PAS are used throughout benzylisoquinoline and pyridine alkaloid biosynthesis. Certain PAS mutations characterize the enzymes found in aspidosperma-alkaloid– and iboga-alkaloid–producing plant clades (Fig. 3C). For instance, PAS lacks the His and Cys residues involved in covalent binding of the FAD cofactor (fig. S28). We anticipate that these aspidosperma-associated PAS homologs populate the metabolic pathways for the wide range of aspidosperma alkaloids found in nature. DPAS is a medium-chain alcohol dehydrogenase, a class of enzymes widely used in monoterpene indole alkaloid biosynthesis (14, 18, 19, 24). We hypothesize that CS and TS may retain the hydrolysis function of the putative ancestor hydrolase enzyme (25) to allow formation of dehydrosecodine 9 from dihydroprecondylocarpine acetate 11. In principle, the formation of tabersonine 2 and catharanthine 3 is formed via two different modes of cyclization, and dehydrosecodine 9 can undergo two distinct Diels-Alder reactions (26) to form either catharanthine 3 or tabersonine 2 (Fig. 2).

Here we report the discovery of two enzymes—PAS and DPAS—along with the discovery of the catalytic function of two other enzymes—CS and TS (11)—that convert stemmadenine acetate 7 to tabersonine 2 and catharanthine 3. Chemical investigations of this system (68, 15), coupled with plant DNA sequence data, enabled discovery of the last enzymes responsible for the construction of the tabersonine 2 or catharanthine 3 scaffolds. With the biosynthesis of stemmadenine acetate 7 (11), this completes the biosynthetic pathway for vindoline 4 and catharanthine 3, compounds that can be used to semisynthetically prepare vinblastine. These discoveries allow the prospect of heterologous production of these expensive and valuable compounds in alternative host organisms, providing a new challenge for synthetic biology (27).

Supplementary Materials

www.sciencemag.org/content/360/6394/1235/suppl/DC1

Materials and Methods

Figs. S1 to S28

Tables S1 to S7

References (3059)

Data S1 to S3

References and Notes

Acknowledgments: We thank G. Saalbach of the John Innes Proteomics Centre for proteomics work, F. Kellner and F. Geu-Flores for early silencing results with CS and TS, B. Lichman for discussions, P. O’Connor for Tabernaemontana material, and R. Hughes and M. Franceschetti for preparing the modified TRBO vector. We also thank MRC PPU Reagents and Services for PAS expression in insect cells. Funding: This work was supported by grants from the European Research Council (311363), BBSRC (BB/J004561/1) (S.E.O.), and the Région Centre-Val de Loire, France (CatharSIS grant and BioPROPHARM project–ARD2020) (V.C.). J.F. acknowledges DFG postdoctoral funding (FR 3720/1-1) and T.-T.T.D. (ALTF 239-2015) and S.C.F. (ALTF 846-2016) acknowledge EMBO Long-Term Fellowships. K.K. acknowledges the French Embassy in Greece for a SSHN grant. R.M.E.P. was supported by a Ph.D. studentship from BBSRC. Author contributions: L.C. characterized all proteins, purified substrates, and coordinated all experiments; J.F. performed experiments with PAS and developed chemical mechanisms and synthetic strategy; S.C.F. helped with candidate selection and VIGS and performed the quantitative polymerase chain reaction experiments; K.C. performed all semisynthetic transformations; R.M.E.P. performed initial cloning and characterization of PAS; T.-D.N. expressed PAS in P. pastoris; T.-T.T.D. performed reconstitution in N. benthamiana; B.A. performed cloning and silencing experiments; D.M.J. helped to identify PAS and designed initial silencing constructs; I.J.C.V. purified stemmadenine; I.S.T.C., K.K., T.D.d.B., and V.C. performed localization and interaction studies; L.C. and S.E.O. wrote the manuscript; and S.C.F., J.F., K.C., and V.C. revised the manuscript. Competing interests: A patent on PAS and DPAS is currently being filed. Data and materials availability: Sequence data for PAS (MH213134), DPAS (KU865331), CS (MF770512), and TS (MF770513) are deposited in GenBank and are also provided in the supplementary materials. Transcriptome data used in this study have already been published (see supplementary materials and http://medicinalplantgenomics.msu.edu/). Cell lines used in this study will be made available upon request. Data supporting the findings of this study are available within the article and the supplementary materials.
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