Bifunctional Cytochrome P450 Enzymes Involved in Camptothecin

Letters Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Bifunctional Cytochrome P450 Enzymes Involved in Camptothecin Biosynthesis Yun Yang,†,§ Wei Li,† Jing Pang,†,§ Liangzhen Jiang,† Xixing Qu,† Xiang Pu,† Guolin Zhang,† and Yinggang Luo*,†,‡ †

Center for Natural Products Research, Chinese Academy of Sciences, Chengdu Institute of Biology, 9 Section 4, Renmin Road South, Chengdu 610041, China ‡ State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China § University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China

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S Supporting Information *

ABSTRACT: Camptothecin (CAM) is a well-known, complex, plant-derived antitumor monoterpenoid indole alkaloid (MIA). Featuring a unique pentacyclic pyrroloquinoline scaffold, CAM is biosynthetically distinct from the other known MIAs, such as antitumor vincristine and vinblastine. Herein, CaCYP72A565 and CaCYP72A610 enzymes involved in the biosynthesis of the monoterpenoid moiety of CAM were cloned from CAM-producing Camptotheca acuminata. Heterologous overexpression and functional characterization assays showed that CaCYP72As catalyzes two consecutive reactions, the stereoselective hydroxylation at C-7 of 7deoxyloganic acid and the subsequent carbon−carbon (C−C) bond cleavage between C-7 and C-8 of iridoid glucoside, to generate the intramolecular cyclopentane ringopening secoiridoid glucoside. Comparative metabolite profiling analyses suggested that C. acuminata synthesizes loganic acid, secologanic acid, and strictosidinic acid as its MIA carboxylic acid intermediates. CaCYP72As are novel bifunctional enzymes that catalyze stereoselective hydroxylation and subsequent C−C bond cleavage reactions to give a ring-opening product with two functional groups, an aldehyde and a double bond.

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small-cell lung and refractory ovarian cancers.12−14 As a distinct pyrroloquinoline-containing MIA, CAM was proposed to biologically synthesize through a modified MIA pathway in plants (Figure 1).15−17 CYPs have been proposed to catalyze many biochemical conversion steps to establish the distinctive pentacyclic pyrroloquinoline MIA.15−17 In the course of the investigations of CAM biosynthesis, we analyzed the public transcriptome data sets from Camptotheca acuminata,18,19 cloned CaCYP72As, and compared them with the functionally characterized CrSLSs (CYP72A1 and its isoform),6,7 the secologanin synthase responsible for the specific C−C bond cleavage conversion of loganin into secologanin (Figure 1B) in Catharanthus roseus (Supporting Information (SI), Figures S1−S5). The CaCYP72As show high amino acid residue sequence identity (>60%) with CrSLSs (Figure S3). Phylogenetic analyses revealed that two CaCYP72As were clustered into one clade with CrSLSs (Figure S4), indicating that they may catalyze the oxidative cyclopentane ring opening of iridoid glucosides to produce secoiridoid glucosides. The CaCYP72As were individually

ytochrome P450 monooxygenases (CYPs), haem-containing enzymes, are the largest superfamily of enzymes that play pivotal roles in metabolism and catabolism in all kingdoms of life.1−5 Generally, CYPs catalyze regio- and stereoselective monooxygenation/hydroxylation, and they participate in many biochemical pathways to produce an immense chemical diversity of species-specific natural products in secondary metabolism.1,2 Some CYPs have been shown to catalyze unique reactions, such as the oxidative rearrangement of carbon skeletons, methylenedioxy bridge formation, phenol coupling, and oxidative carbon−carbon (C−C) bond cleavage.1−4 For instance, CYP72A1 and its isoform from the vincristine- and vinblastine-producing Catharanthus roseus6,7 were functionally proven to catalyze a unique C−C bond cleavage reaction in the cyclopentane ring of loganin to give the ring-opening product secologanin, which is known to be a key intermediate for many pharmaceutically important natural products, such as monoterpenoid indole alkaloids (MIAs), ipecacuanha alkaloids, and secoiridoid glucosides.8,9 Featuring a unique pentacyclic pyrroloquinoline scaffold, camptothecin (CAM, Figure 1) is a well-known plant-derived complex antitumor MIA.10 As unique DNA topoisomerase I inhibitors,11 the CAM-derived topotecan and irinotecan have been approved by the U.S. Food and Drug Administration as antitumor drugs for use against many types of tumors, such as © XXXX American Chemical Society

Received: December 29, 2018 Accepted: May 21, 2019

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DOI: 10.1021/acschembio.8b01124 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Letters

ACS Chemical Biology

Figure 1. Putative biosynthetic pathway for MIAs. Biosynthetic pathway for the monoterpenoid moieties of CAM (A) and of the MIAs in Catharanthus roseus (B). (C) The Pictet−Spengler condensation reaction between tryptamine and the monoterpenoid and the subsequent biochemical modifications to generate MIAs. Enzyme names in bold represent enzymes that have been functionally characterized. Path a, original pathway; path b, recently modified pathway; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; GPP, geranyl diphosphate; GPPS, GPP synthase; GES, geraniol synthase; G10H, geraniol 10-hydroxylase; 10HGO, 10-hydroxygeraniol oxidase; IS, iridodial synthase; 7-DLS, 7deoxyloganetic acid synthase; 7-DLGT, 7-deoxyloganetic acid glucosyltransferase; DL7H, 7-deoxyloganic acid-7-hydroxylase; LAMT, loganic acid O-methyltransferase; SLS, secologanin synthase; TDC, tryptophan decarboxylase; STR, strictosidine synthase; STRAS, strictosidinic acid synthase.

overexpressed (Figure S6) in the Saccharomyces cerevisiae WAT11 strain, which overexpresses the Arabidopsis thaliana NADPH P450 reductase 1.20 The yeast cells/microsomes overexpressing the pYES2-CT empty vector or the pYES2-CTCaCYP72As were incubated with NADPH and loganin. The HPLC-DAD (Figure S7A) and HPLC-DAD-HRMS (Figure 2A) analyses showed that both Caa_locus_1905 and Caa_locus_133 catalyze the conversion of loganin into a new product. The enzymatically converted product showed an identical HPLC retention time (panels II and IV, Figure 2A; panels III and V, Figure S7A) and an identical UV spectrum (panels I and II, Figure S8A) to those of the standard secologanin (panel V, Figure 2A; panel VI, Figure S7A; panel III, Figure S8A). HRESIMS of the product and its fragmentation pattern (panels I and II, Figure S8D) are in perfect accordance with that of the standard secologanin (Figure 2A; Figure S8C). Caa_locus_1905 and Caa_locus_133 displayed intramolecular oxidative C−C bond cleavage activity and were named CYP72A565 and

CYP72A610, respectively, by the CYP nomenclature committee.21 As positive experimental controls, CrSLSs were cloned, overexpressed, and characterized by following the abovedescribed experimental procedures. Because path b (Figure 1) may be present in C. acuminata,17 we tested whether the CaCYP72As can catalyze the conversion of loganic acid into secologanic acid. The enzymatic assays were performed as described above using loganic acid to replace loganin. The HPLC-DAD (Figure S7B) and HPLCDAD-HRMS (Figure 2B) analyses showed that both CaCYP72A565 and CaCYP72A610 catalyze the conversion of loganic acid into a new product. The enzymatically transformed product showed an identical HPLC retention time (panels II and IV, Figure 2B; panels III and V, Figure S7B) and an identical UV spectrum (panels I and II, Figure S8B) to those of the standard secologanic acid (panel V, Figure 2B; panel VI, Figure S7B; panel III, Figure S8B). HRESIMS of the product and its fragmentation pattern (panels I and II, Figure S8F) are in perfect accordance with that of the standard B


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Figure 2. Catalytic oxidative C−C bond cleavage of iridoid glucosides to generate secoiridoid glucosides by CaCYP72As. (A) Relative intensenties of the extracted ion chromatogram (EIC) traces from HPLC-DAD-HRESIMS (positive ionization mode) of the microsomal enzymatic reactions using loganin as a substrate. Standard loganin (blue, [M + Na]+ m/z 413.1419) and secologanin (red, [M + Na]+ m/z 411.1273; panel V). (B) HPLC-DAD-HRMS (positive ionization mode) EICs of the microsomal enzymatic reactions using loganic acid as a substrate. Standard loganic acid (blue, [M + Na]+ m/ z 399.1265) and secologanic acid (red, [M + Na]+ m/z 397.1111; panel V). Panel I, boiled CaCYP72A610; panel II, CaCYP72A610; panel III, boiled CaCYP72A565; panel IV, CaCYP72A565.

Figure 3. Catalytic hydroxylation of 7-deoxyloganic acid to form loganic acid by CaCYP72As. HPLC-DAD-HRESIMS (negative ionization mode) EICs of the microsomal enzymatic reactions using 7-deoxyloganic acid as a substrate. Standard 7-deoxyloganic acid (green, [M − H]− m/z 359.1342), loganic acid (blue, [M − H]− m/z 375.1299), and secologanic acid (red, [M − H]− m/z 373.1143; panel V). Panel I, boiled CaCYP72A610; panel II, CaCYP72A610; panel III, boiled CaCYP72A565; panel IV, CaCYP72A565.

secologanic acid (Figure 2B; Figure S8E). However, CrSLSs could not catalyze the above-mentioned conversion reaction. Phylogenetic analyses (Figure S4) showed that the CaCYP72As are close to CrDL7H (CYP72A224), the 7deoxyloganic acid 7-hydoxylase involved in MIA biosynthesis in Catharanthus roseus (Figure 1B),22 which implied that the CaCYP72As may catalyze the regio- and stereoselective hydroxylation of 7-deoxyloganic acid to give loganic acid. The enzymatic assays were performed as described above using 7-deoxyloganic acid as the substrate. The HPLC-DAD (Figure S9) and HPLC-DAD-HRMS (Figure 3) analyses showed that both CaCYP72A565 and CaCYP72A610 catalyze the conversion of 7-deoxyloganic acid into loganic acid. The enzymatically catalyzed product showed an identical HPLC retention time (panels II and IV, Figure 3; panels III and V, Figure S9A) to that of the standard loganic acid (panel V, Figure 3; panel VI, Figure S9A). UPLC-DAD-HRESIMS of the product and its fragmentation pattern (panel II, Figure S9B; panels I and II, Figure S9D) are in perfect accordance with that of the standard loganic acid (Figure 3; Figure S9C). From the reaction mixtures of the CaCYP72A-catalyzed hydroxylation of 7-deoxyloganic acid, a small product peak showed an identical HPLC retention time (panels II and IV, Figure 3; panels III and V, Figure S9A) to that of the secologanic acid standard (panel V, Figure 3; panel VI, Figure S9A). UPLC-DAD-HRESIMS of the product and its fragmentation ion (panel I, Figure S9B; panels I and II, Figure

S9F) are in perfect accordance with that of the standard secologanic acid (Figure 3; Figure S9E). The results indicate that the CaCYP72As catalyze the regio- and stereoselective hydroxylation of 7-deoxyloganic acid to loganic acid and the subsequent oxidative cyclopentane ring-opening of loganic acid to generate secologanic acid. To validate this conclusion, authentic loganic acid was added to the CaCYP72A-catalyzed 1 h reaction mixtures using 7-deoxyloganic acid as the substrate, and the catalytic conversion reaction was continued for an additional 2 h (panel III, Figure 4; panel IV, Figure S10). Comparing the inactivated CaCYP72A-catalyzed reactions (panel I, Figure S10) with the enzymatic reactions, a small amount of secologanic acid could be detected in the 1 h reaction mixture (panel I, Figure 4; panel II, Figure S10). The amount of secologanic acid increased clearly in the modified 3 h reaction (panel III, Figure 4; panel IV, Figure S10) compared with the normal 3 h reaction (panel II, Figure 4; panel III, Figure S10). Thus, CaCYP72As catalyze consecutive regioand stereoselective hydroxylation and further intramolecular oxidative C−C bond cleavage to generate the cyclopentane ring-opening product with two functional groups, an aldehyde and a double bond. Preliminary investigations of the consecutive reactions implied that the CaCYP72A-catalyzed C−C cleavage activity C


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substrate since it could not be obtained commercially or be derived from 7-deoxyloganic acid via methylation due to the limited amount of 7-deoxyloganic acid. None of these substrate analogues could be converted into the corresponding ringopening products by the CaCYP72As. Thus, for catalytic oxidative C−C bond cleavage, CaCYP72As have strict substrate recognition similar to that of CrSLSs.6,7 CrSLSs cannot catalyze the hydroxylation of 7-deoxyloganic acid but do catalyze a further oxidation of secologanin to generate secoxyloganin. 7 Secoxyloganin, an acidic derivative of secologanin, cannot be condensed with tryptamine by strictosidine synthase, which is ascribed to the lack of the aldehyde functional group.7 The microsomal SLS from cell suspension cultures of Lonicera japonica was shown to have strict substrate specificity, accepting only loganin as a substrate.23 A few bifunctional CYPs have been shown to catalyze both oxidation and unique reactions (SI). However, CaCYP72As are novel bifunctional enzymes that catalyze stereoselective hydroxylation and subsequent intramolecular cyclopentane ring opening through C−C cleavage to give one product with two functional groups, an aldehyde and a double bond. The quantitative real-time PCR analyses showed that both CaCYP72A565 and CaCYP72A610 genes were widely expressed in different tissues of C. acuminata (Figure S15). The accumulation of secologanic acid is positively correlated with the transcription levels of CaCYP72As within different plant tissues (Figure S15). The CAM content in the leaves is positively associated with the transcription levels of the CaCYP72As. In the stems, CAM was found at slightly lower levels than in the leaves, while the stems showed the lowest transcriptional levels of CaCYP72As. The seemingly contradictory results suggest that the leaves are the biosynthetic compartment for CAM and a portion of freshly synthesized CAM is transported to the stems and stored (Figure S15), since the biosynthesis of MIA is thought to be compartmentalized in different plant tissues.13 Five different plant tissues, including roots (SR), stems (SS), and leaves (SL) from seedlings and young leaves (YL) and flower buds (FB) from wild mature C. acuminata, were collected and extracted by two solvent systems to obtain the putative intermediates of the CAM biosynthesis pathway in C. acuminata.17,25 The freshly prepared extracts were immediately subjected to HPLC-DAD and UPLC-HRESIMS analyses (Figure S16). Ten putative biosynthetic intermediates, CAM, and 10-hydroxycamptothecin were detected and identified by comparing the HPLC retention times, UV spectra, and accurate molecular ions observed in the UPLC-HRESIMS mass spectra with those of the standards and/or the proposed biosynthetic intermediates (Table S2). Tryptamine, secologanic acid, and strictosidinic acid were detected in all plant

Figure 4. Consecutive conversion reactions from 7-deoxyloganic acid to secologanic acid via loganic acid catalyzed by CaCYP72A610 (A) and CaCYP72A565 (B). HPLC-DAD-HRESIMS (negative ionization mode) EIC analyses of the microsomal enzymatic reactions using 7deoxyloganic acid as a substrate. Standard 7-deoxyloganic acid (green, [M − H]− m/z 359.1342), loganic acid (blue, [M − H]− m/z 375.1299), and secologanic acid (red, [M − H]− m/z 373.1143; panels IV). Panel I, 1 h reaction; panel II, 3 h reaction; panel III, an additional 1 mM of loganic acid was added to the reaction mixtures of the whole reaction at 1 h and then the reaction was continued for 2 more hours.

is a rate-limiting step (Figures 3 and 4; Figures S9 and S10). The protein overexpression time, reaction buffer pH, temperature, time, NADPH, and substrate concentration were optimized for the catalytic oxidative C−C bond cleavage catalyzed by CaCYP72As (Figure S11). The steady kinetic parameters suggested that CaCYP72A565 shows a slightly higher catalytic efficiency than CaCYP72A610 (Table 1, Figure S12). To investigate the substrate scope of the CaCYP72As, nine iridoid and secoiridoid glucosides, namely, 8-Oacetylharpagide, harpagide, geniposide, geniposidic acid, morroniside, sweroside, sanzhiside methyl, sanzhiside, and verbenalin (Figure S13), were tested for potential catalysis by the CaCYP72As. HPLC-DAD, HRESIMS, and NMR data interpretation revealed that geniposidic acid and its methyl ester geniposide were hydroxylated at C-6β by the CaCYP72As to give scandoside and its methyl ester, respectively (Figure S14), indicating that the CaCYP72As catalyzed stereoselective β-hydroxylation toward a less hindered cyclopentane moiety of iridoid glucosides. However, the recombinant CrDL7H can accept only 7-deoxyloganic acid as a substrate.22 It should be noted that we did not test 7deoxyloganin, the methyl ester of 7-deoxyloganic acid, as a

Table 1. Kinetic Parameters of CaCYP72As with Different Substrates enzyme

substrate

CaCYP72A610

7-deoxyloganic acid loganic acid loganin 7-deoxyloganic acid loganic acid loganin

CaCYP72A565

Kma 0.33 1.78 1.10 0.33 1.50 2.29

± ± ± ± ± ±

0.07 0.63 0.10 0.07 0.48 0.17

Vmaxb

Kcatc

Kcat/Km

± ± ± ± ± ±

0.09 0.04 0.03 0.21 0.08 0.06

0.28 0.02 0.026 0.63 0.05 0.025

0.29 0.14 0.09 0.67 0.25 0.18

0.02 0.04 0.01 0.11 0.04 0.01

Km, mM. bVmax, μM/min. cKcat, μM/min/mM enzyme. All experimental values represent the means of three replicates ± standard deviation.

a

D


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ACS Chemical Biology tissues. Loganic acid was detected in the SL, YL, and FB tissues. As expected, C. acuminata accumulated loganic acid, secologanic acid, and strictosidinic acid but lacked detectable levels of the corresponding methyl ester derivatives loganin, secologanin, and strictosidine. The monoterpenoid intermediates geraniol, 10-hydroxygeraniol, and 7-deoxyloganic acid could not be detected in any of the five plant tissues, which may be ascribed to their instability and volatility. The biosynthetic intermediates of the downstream pathway of CAM,17,24−27 strictosamide and its ketolactam and epoxide, pumiloside, and deoxypumiloside, were identified in all five plant tissues. CAM and 10-hydroxycamptothecin were accumulated in all plant tissues. The comparative metabolomics analyses (Figure S16, Table S2) suggested that C. acuminata might follow an alternative secoiridoid glucoside pathway with carboxylic acid intermediates to strictosidinic acid (path b, Figure 1), which is highly consistent with the results of the latest report.17 However, in the best studied MIA-producing plant Catharanthus roseus, the methyl-esterified intermediates, not the acidic intermediates, have been proven to be involved in MIA biosynthesis.24,28 The results presented here and the previous report17 implied that CAM biosynthesis may be distinct from that of other MIAs. However, the methylesterified intermediate pathway may not be ruled out, since the enzymatic activity assays indicated that the CaCYP72As can catalyze the conversion of loganin into secologanin (Figure 2A; Figures S7, S8, S10, and S11), and we could not clarify whether 7-deoxyloganin can be hydroxylated by CaCYP72As at the moment due to the limited amounts of reagents. More studies, such as molecular cloning and functional characterization of strictosidinic acid synthase (Figure 1), are in progress to uncover the biosynthetic mysteries of CAM, which will be fundamental to developing novel biological platforms to produce CAM efficiently. In summary, we mined the transcriptome data of CAMproducing C. acuminata and cloned candidate CYP72As for the biosynthesis of the monoterpenoid moiety of CAM. CaCYP72A565 and CaCYP72A610 were shown to identically catalyze consecutive reactions, the stereoselective hydroxylation at C-7 of 7-deoxyloganic acid, and the subsequent C−C cleavage between C-7 and C-8 of iridoid glucoside, to generate the intramolecular cyclopentane ring-opening secoiridoid glucoside. Comparative metabolite profiling analyses revealed that C. acuminata synthesizes loganic acid, secologanic acid, and strictosidinic acid as its carboxylic acid intermediates. CaCYP72As are bifunctional CYP enzymes that catalyze stereoselective hydroxylation and subsequent C−C cleavage reactions to give a ring-opening product with two functional groups, an aldehyde and a double bond.

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METHODS

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ASSOCIATED CONTENT

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previously reported bifunctional CYPs, supplementary figures, references, and tables (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yinggang Luo: 0000-0002-8647-0948 Author Contributions

Y.L. conceived and designed the study. Y.Y., X.Q., X.P., and L.J. mined the transcriptome data and cloned the candidate CYPs. Y.Y., W.L., J.P., X.Q., and X.P. performed the recombinant plasmids construction, heterologous expression, enzymatic activity assay, product characterization, and kinetics assay. Y.Y. and X.P. carried out the metabolomics analysis. Y.L., Y.Y., and G.Z. analyzed the data, and Y.L., Y.Y., and G.Z. wrote the manuscript. Funding

This work was supported in part by the 21172216 Project from the National Natural Science Foundation of China, the ZSTH003 Project from the Chinese Academy of Sciences, and the Applied and Basic Research Program of Sichuan Province (2015JY0058). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank D. Nelson at University of Tennessee, USA, for naming the CYPs according to the standardized CYP nomenclature system. We thank T. Xia at Anhui Agricultural University, China, for kindly supplying the S. cerevisiae WAT11 strain. We thank L. Zhou at Northwest A and F University, China, for kindly supplying 7-deoxyloganic acid. We thank S. Song at Shenyang Pharmaceutical University, China, for kindly supplying secologanic acid and secoxyloganin.

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REFERENCES

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General Experimental Procedures. A detailed description of general experimental procedures is given in the Supporting Information.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b01124. Experimental procedures, supplementary results, including transcriptome data mining, molecular cloning, and E


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Bifunctional Cytochrome P450 Enzymes Involved in Camptothecin

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