Bifunctional Cytochrome P450 Enzymes Involved in Camptothecin Biosynthesis

Abstract

Camptothecin (CAM) is a well-known complex plant-derived antitumour monoterpenoid indole alkaloid (MIA). Featuring a unique pentacyclic pyrroloquinoline scaffold, CAM is biosynthetically distinct from the other known MIAs, such as antitumour vincristine and vinblastine. Herein, CaCYP72A565 and CaCYP72A610 enzymes involved in the biosynthesis of monoterpenoid moiety of CAM were cloned from CAM-producing Camptotheca acuminata. Heterologous overexpression and functional characterization assays showed that CaCYP72As catalyse two consecutive reactions, the stereoselective hydroxylation at C-7 of 7-deoxyloganic acid and the subsequent carbon-carbon (C-C) bond cleavage between C-7 and C-8 of iridoid glucoside, to generate the intramolecular cyclopentane ring-opening 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 catalyse stereoselective hydroxylation and subsequent C-C bond cleavage reactions to give ring-opening product with two functional groups, an aldehyde and a double bond.

Full text

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
*
SSupporting 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 7-
deoxyloganic acid and the subsequent carbon−carbon (C−C) bond cleavage between
C-7 and C-8 of iridoid glucoside, to generate the intramolecular cyclopentane ring-
opening 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.
Cytochrome P450 monooxygenases (CYPs), haem-con-
taining 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 cleav-
age.
1−4
For instance, CYP72A1 and its isoform from the
vincristine- and vinblastine-producing Catharanthus roseus
6,7
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
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
specificC−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
Received: December 29, 2018
Accepted: May 21, 2019
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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-CT-
CaCYP72As 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_lo-
cus_133 displayed intramolecular oxidative C−Cbond
cleavage activity and were named CYP72A565 and
CYP72A610, respectively, by the CYP nomenclature commit-
tee.
21
As positive experimental controls, CrSLSs were cloned,
overexpressed, and characterized by following the above-
described 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 HPLC-
DAD-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
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, 7-
deoxyloganetic 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.
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B

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 7-
deoxyloganic 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 con-
version 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 reac-
tions (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 regio-
and 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
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 micro-
somal enzymatic reactions using loganin as a substrate. Standard
loganin (blue, [M + Na]+m/z413.1419) and secologanin (red, [M +
Na]+m/z411.1273; 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/
z399.1265) and secologanic acid (red, [M + Na]+m/z397.1111;
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/z359.1342), loganic acid (blue, [M −H]−m/z
375.1299), and secologanic acid (red, [M −H]−m/z373.1143; panel
V). Panel I, boiled CaCYP72A610; panel II, CaCYP72A610; panel
III, boiled CaCYP72A565; panel IV, CaCYP72A565.
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C

is a rate-limiting step (Figures 3 and 4;Figures S9 and S10).
The protein overexpression time, reaction buffer pH, temper-
ature, 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 secoiridoidglucosides,namely,8-O-
acetylharpagide, 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-6βby 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 7-
deoxyloganin, the methyl ester of 7-deoxyloganic acid, as a
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 ring-
opening 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 ex-
pressed 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 contra-
dictory 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 compartmen-
talized 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, secolo-
ganic 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 7-
deoxyloganic acid as a substrate. Standard 7-deoxyloganic acid (green,
[M −H]−m/z359.1342), loganic acid (blue, [M −H]−m/z
375.1299), and secologanic acid (red, [M −H]−m/z373.1143;
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.
Table 1. Kinetic Parameters of CaCYP72As with Different Substrates
enzyme substrate Km
a
Vmax
b
Kcat
c
Kcat/Km
CaCYP72A610 7-deoxyloganic acid 0.33 ±0.07 0.29 ±0.02 0.09 0.28
loganic acid 1.78 ±0.63 0.14 ±0.04 0.04 0.02
loganin 1.10 ±0.10 0.09 ±0.01 0.03 0.026
CaCYP72A565 7-deoxyloganic acid 0.33 ±0.07 0.67 ±0.11 0.21 0.63
loganic acid 1.50 ±0.48 0.25 ±0.04 0.08 0.05
loganin 2.29 ±0.17 0.18 ±0.01 0.06 0.025
a
Km, mM.
b
Vmax,μM/min.
c
Kcat,μM/min/mM enzyme. All experimental values represent the means of three replicates ±standard deviation.
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D

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 intermedi-
ates 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 metab-
olomics 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 report
17
implied that CAM biosynthesis
may be distinct from that of other MIAs. However, the methyl-
esterified 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 character-
ization 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 CAM-
producing 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 hydroxyla-
tion 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.
■METHODS
General Experimental Procedures. A detailed description of
general experimental procedures is given in the Supporting
Information.
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.8b01124.
Experimental procedures, supplementary results, includ-
ing transcriptome data mining, molecular cloning, and
previously reported bifunctional CYPs, supplementary
figures, references, and tables (PDF)
■AUTHOR INFORMATION
Corresponding Author
*E-mail: yinggluo@cib.ac.cn.
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 ZSTH-
003 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.
■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.
■REFERENCES
(1) Hamberger, B., and Bak, S. (2013) Plant P450s as versatile
drivers for evolution of species-specific chemical diversity. Philos.
Trans. R. Soc., B 368, 20120426.
(2) Ghosh, S. (2017) Triterpene structural diversification by plant
cytochrome P450 enzymes. Front. Plant Sci. 8, 1886.
(3) Mizutani, M., and Sato, F. (2011) Unusual P450 reactions in
plant secondary metabolism. Arch. Biochem. Biophys. 507, 194−203.
(4) Guengerich, F. P., and Munro, A. W. (2013) Unusual
cytochrome P450 enzymes and reactions. J. Biol. Chem. 288,
17065−17073.
(5) Guengerich, F. P., and Yoshimoto, F. K. (2018) Formation and
cleavage of C-C bonds by enzymatic oxidation-reduction reactions.
Chem. Rev. 118, 6573−6655.
(6) Irmler, S., Schröder, G., St-Pierre, B., Crouch, N. P., Hotze, M.,
Schmidt, J., Strack, D., Matern, U., and Schröder, J. (2000) Indole
alkaloid biosynthesis in Catharanthusroseus: new enzyme activities and
identification of cytochrome P450 CYP72A1 as secologanin synthase.
Plant J. 24, 797−804.
(7) Dugéde Bernonville, T., Foureau, E., Parage, C., Lanoue, A.,
Clastre, M., Londono, M. A., Oudin, A., Houillé
, B., Papon, N.,
Besseau, S., Glé
varec, G., Atehortùa, L., Giglioli-Guivarc’h, N., St-
Pierre, B., De Luca, V., O’Connor, S. E., and Courdavault, V. (2015)
Characterization of a second secologanin synthase isoform producing
both secologaninand secoxyloganin allows enhanced de novo assembly
of a Catharanthusroseus transcriptome. BMC Genomics 16, 619.
(8) Battersby, A. R., and Parry, R. J. (1971) Biosynthesis of the
Ipecac alkaloids and of ipecoside. J. Chem. Soc. D 1971, 901−902.
ACS Chemical Biology Letters
DOI: 10.1021/acschembio.8b01124
ACS Chem. Biol. XXXX, XXX, XXX−XXX
E

(9) Inouye, H., Ueda, S., Inoue, K., and Takeda, Y. (1974) Studies
on monoterpene glucosides and related natural products. XXIII.
Biosynthesis of the secoiridoid glucosides, gentiopicroside, morroni-
side, oleuropein, and jasminin. Chem. Pharm. Bull. 22, 676−686.
(10) Wall, M. E., Wani, M. C., Cook, C. E., Palmer, K. H., McPhail,
A. T., and Sim, G. A. (1966) Plant antitumor agents. I. The isolation
and structure of camptothecin, a novel alkaloidal leukemia and tumor
inhibitor from Camptothecaacuminata.J. Am. Chem. Soc. 88, 3888−
3890.
(11) Hsiang, Y. H., Hertzberg, R., Hecht, S., and Liu, L. (1985)
Camptothecin induces protein-linked DNA breaks via mammalian
DNA topoisomerase I. J. Biol. Chem. 260, 14873−14878.
(12) Demain, A. L., and Vaishnav, P. (2011) Natural products for
cancer chemotherapy. Microb. Biotechnol. 4, 687−699.
(13) Sirikantaramas, S., Asano, T., Sudo, H., Yamazaki, M., and
Saito, K. (2007) Camptothecin: therapeutic potential and biotechnol-
ogy. Curr. Pharm. Biotechnol. 8, 196−202.
(14) Thomas, C. J., Rahier, N. J., and Hecht, S. M. (2004)
Camptothecin: current perspectives. Bioorg. Med. Chem. 12, 1585−
1604.
(15) Kai, G., Wu, C., Gen, L., Zhang, L., Cui, L., and Ni, X. (2015)
Biosynthesis and biotechnological production of anti-cancer drug
Camptothecin. Phytochem. Rev. 14, 525−539.
(16) Qu, X., Pu, X., Chen, F., Yang, Y., Yang, L., Zhang, G., and Luo,
Y. (2015) Molecular cloning, heterologous expression, and functional
characterization of an NADPH-cytochrome P450 reductase gene from
Camptothecaacuminata, a camptothecin-producing plant. PLoS One
10, No. e0135397.
(17) Sadre, R., Magallanes-Lundback, M., Pradhan, S., Salim, V.,
Mesberg, A., Jones, A. D., and DellaPenna, D. (2016) Metabolite
diversity in alkaloid biosynthesis: a multilane (diastereomer) highway
for camptothecin synthesis in Camptothecaacuminata.Plant Cell 28,
1926−1944.
(18) Sun, Y., Luo, H., Li, Y., Sun, C., Song, J., Niu, Y., Zhu, Y., Dong,
L., Lv, A., Tramontano, E., and Chen, S. (2011) Pyrosequencing of
the Camptothecaacuminata transcriptome reveals putative genes
involved in camptothecin biosynthesis and transport. BMC Genomics
12, 533.
(19) Góngora-Castillo, E., Childs, K. L., Fedewa, G., Hamilton, J. P.,
Liscombe, D. K., Magallanes-Lundback, M., Mandadi, K. K., Nims, E.,
Runguphan, W., Vaillancourt, B., Varbanova-Herde, M., DellaPenna,
D., McKnight, T. D., O'Connor, S., and Buell, C. B. (2012)
Development of transcriptomic resources for interrogating the
biosynthesis of monoterpene indole alkaloids in medicinal plant
species. PLoS One 7, No. e52506.
(20) Pompon, D., Louerat, B., Bronine, A., and Urban, P. (1996)
Yeast expression of animal and plant P450 in optimized redox
environments. Methods Enzymol. 272,51−64.
(21) Nelson, D. R. (2006) Cytochrome P450 nomenclature, 2004.
Methods Mol. Biol. 320,1−10.
(22) Salim, V., Yu, F., Altarejos, J., and De Luca, V. (2013) Virus-
induced gene silencing identifies Catharanthusroseus 7-deoxyloganic
acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid
biosynthesis. Plant J. 76, 754−765.
(23) Yamamoto, H., Katano, N., Ooi, A., and Inoue, K. (2000)
Secologanin synthase which catalyzes the oxidative cleavage of loganin
into secologanin is a cytochrome P450. Phytochemistry 53,7−12.
(24) Dugéde Bernonville, T., Clastre, M., Besseau, S., Oudin, A.,
Burlat, V., Glé
varec, G., Lanoue, A., Papon, N., Giglioli-Guivarc’h, N.,
St-Pierre, B., and Courdavault, V. (2015) Phytochemical genomics of
the Madagascar periwinkle: Unravelling the last twists of the alkaloid
engine. Phytochemistry 113,9−23.
(25) Montoro, P., Maldini, M., Piacente, S., Macchia, M., and Pizza,
C. (2010) Metabolite fingerprinting of Camptothecaacuminata and the
HPLC-ESI-MS/MSanalysis of camptothecin and related alkaloids. J.
Pharm. Biomed. Anal. 51, 405−415.
(26) Carte, B. K., DeBrosse, C., Eggleston, D., Hemling, M.,
Mentzer, M., Poehland, B., Troupe, N., Westley, J. W., and Hecht, S.
M. (1990) Isolation and characterization of a presumed biosynthetic
precursor of camptothecin from extracts of Camptothecaacuminata.
Tetrahedron 46, 2747−2760.
(27) Hutchinson, C. R., Heckendorf, A. H., Straughn, J. L., Daddona,
P. E., and Cane, D. E. (1979) Biosynthesis of camptothecin. 3.
Definition of strictosamide as the penultimate biosynthetic precursor
assisted by carbon-13 and deuterium NMR spectroscopy. J. Am.
Chem. Soc. 101, 3358−3369.
(28) O’Connor, S. E., and Maresh, J. J. (2006) Chemistry and
biology of monoterpene indole alkaloid biosynthesis. Nat. Prod. Rep.
23, 532−547.
ACS Chemical Biology Letters
DOI: 10.1021/acschembio.8b01124
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