Diterpene Biosynthesis in Catenulispora acidiphila: On the Mechanism of Catenul-14-en-6-ol Synthase

Article abstract of DOI:10.1002/anie.202014180

A new diterpene synthase from the actinomycete Catenulispora acidiphila was identified and the structures of its products were elucidated, including the absolute configurations by an enantioselective deuteration approach. The mechanism of the cationic terpene cyclisation cascade was deeply studied through the use of isotopically labelled substrates and of substrate analogues with partially blocked reactivity, resulting in derailment products that gave further insights into the intermediates along the cascade. Their chemistry was studied, leading to the biomimetic synthesis of a diterpenoid analogue of a brominated sesquiterpene known from the red seaweed Laurencia microcladia.

Full text of DOI:10.1002/anie.202014180

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Diterpene Biosynthesis in Catenulispora acidiphila: On the
Mechanism of Catenul-14-en-6-ol Synthase
Authors: Geng Li, Yue-Wei Guo, and Jeroen Sidney Dickschat
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Diterpene Biosynthesis in Catenulispora acidiphila: On the
Mechanism of Catenul-14-en-6-ol Synthase
Geng Li,[a,b,c] Yue-Wei Guo*[b,c] and Jeroen S. Dickschat*[a]
Abstract:
A
new diterpene synthase from the actinomycete
labelled substrates[12] or substrate analogs,[13] and DFT
calculations,[6] but the cationic intermediates along the cascade
cannot be observed experimentally. During the past few years
several di- and sesterterpene synthases (DTSs / StTSs) were
identified from all kingdoms of life.[2,12,14-17] As a result of the
multiple reactive double bonds in their substrates geranylgeranyl
(GGPP) and geranylfarnesyl diphosphate (GFPP) the cyclisation
cascades for these enzymes are usually more complex and
interesting to study than the cyclisations of geranyl (GPP) and
farnesyl diphosphate (FPP) by mono- and sesquiterpene
synthases (MTSs / STSs). We envisioned that substrate analogs
with saturated double bonds would allow for interesting insights
into diterpene cyclisation cascades, because they cannot
undergo certain cyclisation steps as observed for GGPP and
could lead to derailment products that may reflect early
cyclisation events. Here we report on the identification of a new
DTS from Catenulispora acidiphila and its mechanistic
interrogation with isotopically labelled probes and with such
GGPP analogs of limited reactivity.
Catenulispora acidiphila was identified and the structures of its
products were elucidated, including the absolute configurations by
an enantioselective deuteration approach. The mechanism of the
cationic terpene cyclisation cascade was deeply studied through the
use of isotopically labelled substrates and of substrate analogs with
partially blocked reactivity, resulting in derailment products that gave
further insights into the intermediates along the cascade. Their
chemistry was studied, leading to the biomimetic synthesis of a
diterpenoid analog of a brominated sesquiterpene known from the
red seaweed Laurencia microcladia.
The fascinating skeletal diversity of terpenes is generated by
terpene synthases (TSs) from a limited number of oligoprenyl
diphosphates.[1-3] These intriguing biocatalysts perform some of
the mechanistically most complex transformations in Nature, yet
the basic principle of TS catalysis is remarkably simple at the
same time. The initiation of terpene cyclisations always
proceeds by substrate ionisation, either through diphosphate
abstraction from or by protonation of the substrate,[4,5] while the
subsequent events are determined by the substrate
conformation that is controlled by the shape of the enzyme’s
hydrophobic active site.[6] After cation formation, a multistep
cascade with cyclisations by intramolecular attack of double
bonds to cationic centres or – rarely – ring openings or
fragmentations by the reverse process,[7] Wagner-Meerwein or
dyotropic rearrangements,[8-10] hydride or proton shifts, attack of
water or alcohol groups to a cation in the formation of terpene
alcohols or ethers, and a terminal deprotonation is promoted.
Intriguingly, all steps are performed with precise stereo-, but
very limited direct enzyme control, that is almost restricted to the
stabilisation of cationic intermediates through cation-
interactions. The overall process results in the installation of
usually polycyclic skeletons with multiple stereogenic centres.
Mechanistic investigations of TSs can be performed by structure
based site-directed mutagenesis,[11] usage of isotopically
Figure 1. Total ion chromatogram of the products obtained from GGPP with
CaCS.
The actinomycete C. acidiphila DSM 44928 caught our interest
to study its TSs, because this strain was found to emit traces of
a volatile diterpene hydrocarbon (Figure S1), suggesting that
one of the TSs encoded in its genome should have DTS activity.
From the two candidate enzymes returned by a BLAST search
one appeared within a phylogenetic tree constructed from 3267
bacterial TS homologs close to several known STSs, while the
second showed close phylogenetic relation to known DTSs and
exhibited all highly conserved motifs expected for a functional
enzyme (Figures S2 and S3), and was thus selected for further
study. Gene cloning and expression followed by incubation of
the purified protein (Figure S4) with GGPP resulted in the
efficient formation of several diterpenes (Figures 1 and S5),
while GPP, FPP and GFPP were not converted. Three major
products obtained from GGPP were isolated by silica gel
chromatography followed by HPLC purification, and their
structures were elucidated by one- and two-dimensional NMR
spectroscopy (Tables S2 – S4, Figures S6 – S26), resulting in
the structures of the main product catenul-14-en-6-ol (1) with a
[a]
[b]
[c]
Geng Li, Prof. Dr. Jeroen S. Dickschat
Kekulé-Institute of Organic Chemistry and Biochemistry
University of Bonn
Gerhard-Domagk-Strasse 1
53121 Bonn, Germany
Email: dickschat@uni-bonn.de
Geng Li, Prof. Dr. Yue-Wei Guo
State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences
555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park
201203, Shanghai, China
Email: ywguo@simm.ac.cn
Geng Li, Prof. Dr. Yue-Wei Guo
University of Chinese Academy of Sciences
No. 19A Yuquan Road, Beijing 100049, China
Supporting information for this article is given via a link at the end of
the document.
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6-6-6 tricyclic system, and two hydrocarbons with the skeleton of
a 6-7-5 tricyclic system, named isocatenula-2,14-diene (2) and
isocatenula-2(6),14-diene (3). This identified the enzyme as
Catenulispora acidiphila Catenul-14-en-6-ol Synthase (CaCS). A
BLAST search and amino acid sequence alignment with the four
closest hits suggests that enzymes with the same function may
be present in a few other actinomycetes (Figure S27).
isotopomers of GPP, FPP or isopentenyl diphosphate (IPP), with
CaCS resulted in the labelled compounds 1 – 3 carrying the 13C-
label in the expected positions from all labelled substrates
(Figure S28, for an example cf. Figure 2). These experiments i)
supported the GGPP fold leading to 1 – 3 and ii) revealed a strict
stereochemical course for the geminal methyl groups C16 and
C17 that did not show any scrambling of 13C-labelling between
these positions.
Figure 2. Labelling experiment with (1-13C)GGPP and CaCS. 13C-NMR
spectra of A) 1, B) 2, C) 3, and D) product mixture obtained from (1-13C)GGPP
with CaCS. Dots represent 13C-labelled carbons.
The 1,3-hydride shift from C1 to C11 was followed by using (R)-
and (S)-(1-2H)IPP,[18] which were used to elongate (7-13C)FPP
with GGPPS to (R)- and (S)-(1-2H,11-13C)GGPP, followed by
cyclisation with CaCS (Figure S29). The hydride shift resulted in
a direct 13C-2H connection in 1 when using (S)-(1-2H)IPP, as
1
indicated by an upfield shifted triplet peak (–0.47 ppm, JC,D
=
19.0 Hz) for C11 in the 13C-NMR, while a singlet with a minor
upfield shift (–0.01 ppm) was obtained with (R)-(1-2H)IPP, in
agreement with a deuterium located two positions away. The
alternative of two sequential 1,2-hydride shifts from A to B was
excluded by incubation of (3-13C,2-2H)GPP[20] and IPP with
GGPPS and CaCS, showing a singlet with an upfield shift (–0.08
ppm) in the 13C-NMR as a result of deuterium bound at a carbon
next to C11, indirectly giving further evidence for the 1,3-hydride
shift. We have previously shown that the analogous hydride shift
occuring in the germacradienyl cation in the biosynthesis of
many sesquiterpenes can be taken as an indicator for the
absolute configurations of the products, as for 10S configuration
the specific migration of the 1-pro-S hydrogen (HS) was
observed, while for 10R configuration the 1-pro-R hydrogen (HR)
was shifted.[19] Along similar lines, the specific migration of HS in
A may be taken as an indicator for 10S configuration and thus
for the absolute configurations of 1 – 3 as shown in Scheme 1
(for confirmation of absolute configurations vide infra).
Scheme 1. Cyclisation cascade from GGPP to the diterpenes 1 – 3 by CaCS.
Carbon numberings of 1 – 3 were assigned so that the origin for each carbon
from GGPP can be followed by same number.
The proposed cyclisation cascade for 1 – 3 (Scheme 1) starts
from GGPP by 1,10-cyclisation to A, followed by a 1,3-hydride
shift to B, 1,14-cyclisation to C and deprotonation to the neutral
intermediate D. Its reprotonation at C3 can induce another 2,7-
cyclisation with attack of water that may be concerted to avoid a
secondary cation as intermediate, yielding 1. Alternatively, the
protonation at C3 of D can induce a 2,6-cyclisation to E that
upon a 1,2-hydride migration to F and deprotonation gives
access to 3. Branching out from F, another 1,2-hydride transfer
to G and deprotonation result in 2. This mechanism was
investigated in
a series of incubation experiments with
isotopically labelled terpene precursors (Table S5). The
conversion of all 20 isotopomers of (13C)GGPP, partly
synthesised as reported previously and partly made accessible
through enzymatic reactions by GGPP synthase (GGPPS) from
Streptomyces cyaneofuscatus[15] from the corresponding
The deprotonation at C14 from C to D was evident from an
experiment with (2-2H)DMAPP[21] that was elongated with IPP
and GGPPS to (14-2H)GGPP, followed by cyclisation through
CaCS. The mass spectra of 1 – 3 (Figure S30) demonstrated
the deprotonation step at C14 from C to D by the observed
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molecular ions at m/z = 290 and 272, respectively. The
reprotonation of D at C3 with an external proton from the
medium was demonstrated by incubation of FPP and (3-
13C)IPP[15] with GGPPS and CaCS in D2O buffer, resulting in the
typical upfield shifted triplet peak in the 13C-NMR of 1 indicating
a direct 13C-2H connection (Figure S31). Both experiments
together also ruled out the hypothetical alternative of an
intramolecular proton transfer from C14 to C3 in C that would
bypass the neutral intermediate D. GC/MS analysis of the
products obtained in the latter experiment simultaneously
demonstrated the loss of the same deuterium in the
deprotonation from G to 2 as taken up in the C3 reprotonation of
D by a molecular ion at m/z = 273 (Figure S32).
migration of the 1-pro-S hydrogen in A, discussed above as a
potential indicator of a 10S configuration, was confirmed.
The 1,2-hydride shift from E to F was demonstrated using the
labeled substrate (2-2H,3-13C)FPP[22] together with IPP, GGPPS
and CaCS to yield (7-13C,7-2H)-2 for which a triplet peak was
observed in the 13C-NMR (Figure S33). Along similar lines, the
1,2-hydride shift from F to G was evident from an upfield shifted
triplet for C6 of 2 from substrates (2-13C)FPP[23] and (S)-(2-
2H)IPP, generated in situ from (2-2H)DMAPP with IDI from
Escherichia coli,[24,25] with GGPPS and CaCS (Figure S34).
Furthermore, GC/MS analysis of the products from the same
experiment demonstrated the loss of deuterium in the
deprotonation from C2 in F to yield 3 by a molecular ion at m/z =
273 (Figure S35). If (2-13C)FPP was replaced by (3-13C)FPP,[23]
an upfield shifted singlet (–0.08 ppm) for C7 occurred in the 13C-
NMR spectrum (Figure S36), which further supported the 1,2-
hydride shift and ruled out a direct 1,3-hydride shift from E to G
in the biosynthesis of 2.
The absolute configurations of 1 – 3 were determined using the
substrate DMAPP together with the stereoselectively deuterated
probes (E)- and (Z)-(4-13C,4-2H)IPP,[26] which were enzymatically
converted with the combination of FPP synthase (FPPS) from S.
coelicolor[27] and GGPPS for higher efficiency into
stereoselectively deuterated and 13C-labelled GGPP (Figures
S37, S39 and S41). This reaction proceeds through a known
stereochemical course with attack at C4 of IPP from the Si
face.[28] Subsequent cyclisation by CaCS yielded labelled 1 – 3
in which the deuterated carbon atoms serve as configurationally
well defined stereogenic centres. The substitution of one of the
two diastereotopic hydrogens by deuterium can be monitored
with high sensitivity through HSQC spectroscopy. As a result of
the 13C-labelling, the signal for the remaining hydrogen is
strongly enhanced, while the signal of the hydrogen replaced by
deuterium is vanished. Together with the assignment of the
relative orientation of the diastereotopic hydrogens with respect
to the naturally present stereogenic centres in 1 – 3 by NOESY
the absolute configurations of all three compounds could be
concluded. Similar experiments were performed with (R)- and
(S)-(1-13C,1-2H)IPP,[25] which were converted with IDI, FPPS,
GGPPS, and CaCS into labelled 1 – 3 (Figures S38, S40 and
S42). Herein, the attack at C1 of prenyl diphosphates is known
to proceed with inversion of configuration.[28] Through these
experiments every methylene group of 1 – 3 (C4, C5, C8, C9,
C12 and C13) was stereoselectively deuterated, and all
experiments simultaneously pointed to the same absolute
configurations as shown in Scheme 1. Moreover, the absolute
configurations as already suspected from the specific 1,3-
Scheme 2. Interception of the diterpene cyclisation by CaCS with 14,15-
dihydro-GGPP. A) Cyclisation mechanism to 4 and its MgSO4 dependent
degradation, B) derivatisation of 4 with N-bromosuccinimide (NBS). Carbon
numberings deviate from that for 5a in the literature and indicate the origin of
each carbon from 14,15-dihydro-GGPP by same number.
To further explore the cyclisation mechanism of CaCS,
incubation experiments with substrate analogs with partially
blocked reactivity were performed. For this purpose, 6,7-dihydro-
GPP was synthesised (Scheme S1), elongated twice with IPP
using GGPPS, and cyclised by CaCS. While the 1,10-cyclisation
to A’ and the subsequent 1,3-hydride shift to B’, the analogs of
the natural intermediates A and B, are still possible, the next
1,14-cyclisation cannot proceed as a consequence of the
saturated C14-C15 bond (Scheme 2). Therefore, we assumed
that this substrate could yield 1,10-cyclised derailment products,
thus giving insights into the early cyclisation events. Extraction
of the products, drying of the organic layer with MgSO4 and
chromatographic purification yielded two compounds that were
characterised by NMR spectroscopy (Tables S6 and S7, Figures
S43 – S56) as biflora-4,10(19)-diene (5), the dihydro-analog of
the natural diterpene biflora-4,10(19),15-triene (5a) from
Cubitermes umbratus soldiers,[29] and biflora-4,10-diene (6).
Their structures supported an initial 1,10-cyclisation, but also
showed an additional 1,6-cyclisation not observed in the native
enzyme products 1 – 3 obtained from GGPP. The cyclisation
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mechanisms for 5 and 6 were investigated by isotopic labelling
experiments with 6,7-dihydro-GPP and all five isotopomers of
(13C)IPP (Table S8), yielding doubly labelled isotopomers of 5
and 6 with GGPPS and CaCS. Similarly, the usage of 10,11-
dihydro-FPP[30] with all five (13C)IPPs, and of synthetic (1-13C)-
and (2-13C)-6,7-dihydro-GPP (Scheme S1) with IPP gave seven
more singly labelled isotopomers of 5 and 6. Their 13C-NMR
spectra (Figure S57) indicated the origin for each carbon in the
We also envisioned that the protonation induced cyclisation of D
to E (Scheme 1) could be blocked by use of 6,7-dihydro-GGPP,
and its derailment products could give further evidence for the
cyclisation mechanism by CaCS. Therefore, 6,7-dihydro-GGPP
was also synthesised (Scheme S3) and enzymatically converted
with CaCS. However, this substrate was not converted efficiently
and despite the usage of 220 mg of substrate only some trace
compounds could be observed by GC/MS (Figure S85).
2
C1–C10 + C19 and C20 portion. Moreover, the JC,C couplings
In summary, the diterpene synthase CaCS from C. acidiphila
was functionally characterised and found to produce the novel 6-
6-6 tricyclic diterpene alcohol catenul-14-en-6-ol (1) and two 6-7-
observed in the double labelling experiments with (1-13C)IPP
(labelling at C1/C5) and with (2-13C)IPP (labelling at C2/C6)
gave further evidence for the 1,6-ring closure in 5 and 6.
5
tricyclic compounds, isocatenula-2,14-diene (2) and
The absolute configurations of 5 and 6 were determined by the
enantioselective labelling strategy using 6,7-dihydro-GPP with
(R)- and (S)-(1-13C,1-2H)IPP and with (E)- and (Z)-(4-13C,4-
isocatenula-2(6),14-diene (3). Extensive labelling studies gave
detailed insights into the cyclisation cascade and the absolute
configurations of all three products. Furthermore, substrate
analogs with a saturated double bond were synthesised and
enzymatically converted, leading for 14,15-dihydro-GGPP to a
series of derailment products that further supported the
mechanistic model for the cyclisation cascade. As substrate
analogs may adopt a different conformation in the enzyme than
the native substrate, the absolute configurations and cyclisation
mechanisms to all derailment products were also investigated by
labelling experiments, demonstrating that all compounds are
formed from a common substrate fold. Some of the obtained
derailment compounds showed structural similarities to known
natural products from termites and soft corals and also allowed
for a deeper understanding of their chemistry. However, the
starting point of our study was the observed diterpene
production by C. acidiphila, and although by the present work a
diterpene synthase with interesting products has been
discovered, none of these diterpenes showed an identical mass
spectrum to the compounds observed in headspace extracts
from C. acidiphila (compare Figures S1 and S5), suggesting that
a different DTS may be responsible for its biosynthesis. Its
discovery will be on the list of our future research.
2H)IPP,
or
(R)-
and
(S)-(1-13C,1-2H)-6,7-dihydro-GPP
(synthesised as in Scheme S2 in high enantiomeric purity,
Figure S58) with IPP (Figures S59 – S64). The experiments with
(R)- and (S)-(1-13C,1-2H)IPP also demonstrated the specific
migration of the 1-pro-S hydrogen from A’ to B’. The absolute
configurations of 5 and 6 are analogous to that of natural 5a.[31]
Compound 5a is also known as a degradation product of
obscuronatin (4a), a known diterpene alcohol from the soft coral
Xenia obscuronata[32] that shares the skeleton with the
hypothetical intermediate 14,15-dihydroobscuronatin (4).
Therefore, we assumed 5 and 6 may likewise be degradation
products of 4, and indeed their formation depended on the
usage of MgSO4 during workup (Figures S65 and S66),
suggesting that the 1,6-cyclisation in 5 and 6 is not under
enzyme control. Their formation can be explained by water
elimination through cations H and I. Workup without drying the
organic extract with MgSO4 allowed the isolation of 4, followed
by structure elucidation through NMR spectroscopy (Table S9,
Figures S67 – S73), revealing the structure of an only 1,10-
cyclised enzyme product and thus further supporting the 1,10-
cyclisation as an early event of CaCS catalysis. Labelling of the
carbons of the core structure strengthened the structural
2
assignment, this time giving no JC,C couplings in the double
Acknowledgements
labelling experiments with 6,7-dihydro-GPP and (1-13C)IPP or (2-
13C)IPP in agreement with the macrocyclic ring, and allowed to
follow the biosynthetic origin of the labelled carbons (Figure
S74). However, the relative configuration of 4 with its distant
stereogenic centres at C3 and C10/C11 could not independently
be determined by NOESY. Therefore, the skeleton was rigidified
in a reaction with NBS, resulting in the formation of 7 for which
the full relative configuration could be determined by NMR-
based structure elucidation (Table S10, Figures S75 – S81).
Assuming that the stereogenic centres in 4 are not affected in
the conversion to 7, these results allowed to deduce the full
relative configuration for 4. Notably, this reaction may have
some biosynthetic significance, as vanadium-dependent
haloperoxidases can catalyse similar transformations, which is
especially important in the marine environment.[33-35] The
brominated sesquiterpene 7a is structurally closely related to 7
and is a known natural product from the seaweed Laurencia
microcladia[36] that potentially arises by such an enzymatic
reaction. The absolute configuration of 4 was determined
through enantioselective deuteration (Figures S82 – S84).
This work was funded by the DFG (DI1536/7-2) and also
partially by the Natural Science foundation of China (81991521
and 81520108028). Geng Li is thankful for funding from the
CAS-DAAD Doctoral Joint Scholarship Program. We thank
Andreas Schneider for HPLC purifications.
Keywords: terpenoids • biosynthesis • enzyme mechanisms •
isotopes • substrate analogs
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10.1002/anie.202014180
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
A new diterpene synthase (CaCS)
from Catenulispora acidiphila and its
products were identified. The enzyme
mechanism was studied by isotopic
labelling experiments and usage of
substrate analogs with blocked
reactivity, resulting in a series of
dereailment products. Their chemistry
was studied, leading to the biomimetic
synthesis of a diterpenoid analog of a
brominated sesquiterpene known from
the red seaweed Laurencia
Geng Li, Yue-Wei Guo* and Jeroen S.
Dickschat*
Page No. – Page No.
Diterpene
Biosynthesis
in
Catenulispora acidiphila: On the
Mechanism of Catenul-14-en-6-ol
Synthase
microcaldia.
This article is protected by copyright. All rights reserved.