Characterisation of three terpene synthases for β-barbatene, β-araneosene and nephthenol from social amoebae

Article abstract of DOI:10.1039/c9cc07681f

The products of three terpene synthases from two social amoebae, Dictyostelium discoideum and Dictyostelium purpureum, were identified, showing sesquiterpene synthase activity for one and diterpene synthase activity for the other two enzymes. Site-directed mutagenesis experiments revealed the importance of a newly identfied highly conserved residue for catalytic activity. For one of the enzyme products, β-araneosene, a bromonium ion induced transannular cyclisation was investigated, yielding the first brominated derivatives of this diterpene.

Full text of DOI:10.1039/c9cc07681f

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Characterisation of three terpene synthases
for b-barbatene, b-araneosene and nephthenol
from social amoebae†
Cite this: Chem. Commun., 2019,
55, 13255
Received 30th September 2019,
Accepted 11th October 2019
c
a
Jan Rinkel,a Tobias G. Kollner,b Feng Chen
and Jeroen S. Dickschat
*
¨
DOI: 10.1039/c9cc07681f
rsc.li/chemcomm
The products of three terpene synthases from two social amoebae, identification of the products of three TPSs from social amoebae,
Dictyostelium discoideum and Dictyostelium purpureum, were identi- one enzyme from D. discoideum and two enzymes from Dictyostelium
fied, showing sesquiterpene synthase activity for one and diterpene purpureum.
synthase activity for the other two enzymes. Site-directed mutagenesis
The product of DdTPS9 from D. discoideum AX4 (accession
experiments revealed the importance of a newly identfied highly con- number XP_642260, gene locus tag DDB_G0278279) was pre-
served residue for catalytic activity. For one of the enzyme products, viously identified by GC/MS as b-barbatene (4, Fig. 1B),14 but
b-araneosene, a bromonium ion induced transannular cyclisation was the absolute configuration remained unknown. For full struc-
investigated, yielding the first brominated derivatives of this diterpene.
tural assignment of the product, the gene was cloned into
pET32a and heterologously expressed in Escherichia coli. The
Terpenes constitute one of the largest and – arguably – structurally purified protein (Fig. S1, ESI†) was incubated with GPP, FPP
and biosynthetically most intriguing classes of natural products. The and GGPP, resulting in one main sesquiterpene product from
whole class of compounds arises from just a few linear precursors FPP (Fig. S2, ESI†), while no products were obtained from the
including geranyl (GPP), farnesyl (FPP) and geranylgeranyl dipho- other two substrates. The isolated sesquiterpene showed iden-
sphate (GGPP) by the action of terpene synthases (TPSs) that can tical NMR data to 4 (Table S2 and Fig. S3–S9, ESI†),17 and the
produce numerous different carbon skeletons of mono-, sesqui- and optical rotation was [a]2D0 = À11.1 (c 0.25, C6D6), establishing
diterpenes in just one enzymatic step. Generally, TPSs catalyse DdTPS9 as (À)-b-barbatene synthase. This compound was first
the substrate ionisation by abstraction of diphosphate (type I and reported from several liverworts18,19 and also occurs in the
UbiA-related TPSs)1–4 or by protonation of an olefine (type II).5 The basidiomycete Piptoporus betulinus,20 while its enantiomer
cyclisation cascade then proceeds via a series of cationic intermedi- (+)-4 is known from the higher plant Meum athamanticum21
ates with typical elementary reactions such as cyclisations by olefin and fungi of the genus Fusarium.22 The isomer a-barbatene is a
addition to the cation, Wagner-Meerwein rearrangements, hydride known side product of trichodiene synthase from Fusarium
transfers, and a final deprotonation or nucleophilic attack of water.6
With respect to type I TPSs, many enzymes from plants,7,8 bacteria9,10
and fungi11–13 have been characterised, while only recently the first
enzymes from eukaryotic soil microorganisms – social amoebae –
have been discovered.14 TPSs with fully characterised products
include the synthases for the sesquiterpenes protoillud-7-ene (1)
and asterisca-2(9),6-diene (2),15 and for the diterpene b-pinacene (3)
from Dictyostelium discoideum (Fig. 1A).16 Here we report on the
a
´
¨
Kekule-Institut fur Organische Chemie und Biochemie, Rheinische Friedrich-
¨
Wilhelms-Universitat Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany.
E-mail: dickschat@uni-bonn.de
b
¨
Max Planck Institute for Chemical Ecology, Hans-Knoll-Straße 8, 07745 Jena,
Germany
c Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive,
Knoxville, TN 37996-4561, USA
Fig. 1 Products of TPSs from social amoebae. (A) TPS products identified
in previous work, (B) products reported in this study.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc07681f
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FPPS and DdTPS9. Product analysis by HSQC spectroscopy
showed specific deuterium incorporation at C10 into H10b
(Fig. S13, ESI†). Individual labelling of the geminal methyl
groups in FPP using (12-13C)-30 and (13-13C)FPP, enzymatically
prepared from (9-13C)GPP,31 established the specific migration
of C12 from D+ to E+ along the cyclisation to 4 (Fig. S14, ESI†).
We have recently investigated the functions of several TPSs
from D. purpureum AX1.32 While this previous work did not
reveal a significant diterpene synthase activity for DpTPS10,
DpTPS11 yielded an unidentified diterpene alcohol. Both
enzymes were reinvestigated here, showing the efficient con-
version into diterpenes that were isolated and identified by
NMR spectroscopy. Specifically, the first studied enzyme
DpTPS10 (accession number AXN72979) was obtained by het-
erologous expression in E. coli (Fig. S1, ESI†) and converted
GGPP into a single diterpene hydrocarbon (Fig. S15, ESI†),
while GPP and FPP were not effectively accepted as substrates.
The diterpene was isolated and identified by NMR spectroscopy
as b-araneosene (5, Table S3 and Fig. S16–S22, ESI†). Its absolute
configuration was established based on the optical rotation of
[a]2D0 = À133 (c 0.64, C6D6), demonstrating that 5 is the same
enantiomer as known from the gorgonian Eunicea laciniata,33
which was confirmed by analogous stereoselective labelling
Scheme 1 Cyclisation mechanism from FPP to 4 by DdTPS9.
sporotrichioides,23 but no barbatene synthase has been reported experiments as described above for 4 (Fig. S23 and S24, ESI†).
so far (Scheme 1).
The optical antipode (+)-5 is known from Phomopsis amygdali34
The absolute configuration of (À)-4 was originally assigned and is a side product of the fusicocca-2,10(14)-diene synthase
by X-ray analysis of a p-bromobenzoate derivative24 and is from this fungus.35 Its isomer d-araneosene is an intermediate of
independently confirmed here. For this purpose, a chemical the dolasta-1(15),8-diene synthase from Colletotrichum gloeospor-
correlation was performed using synthetic stereoselectively ioides,31 but no diterpene synthase for (À)-5 has been reported
deuterated (R)- and (S)-(1-13C,1-2H)isopentenyl diphosphate before. The cyclisation from GGPP to 5 is a straightforward
(IPP)25 in conjunction with isopentenyl diphosphate isomerase reaction involving a consecutive 1,11-10,14 cyclisation to G+ and
(IDI) from E. coli BL2125 and FPP synthase (FPPS) from Strepto- deprotonation (Scheme 2). Labelling experiments with (16-13C)-
myces coelicolor A3(2),15 and (E)- and (Z)-(4-13C,4-2H)IPP26 with and (17-13C)GGPP demonstrated a clearly defined course with
dimethylallyl diphosphate (DMAPP) and FPPS, in order to respect to the fate of the geminal methyl groups (Fig. S25, ESI†).
obtain FPP isotopomers with enantioselective deuterations at
The second investigated TPS from D. purpureum (DpTPS11,
each chain methylene group (Fig. S10 and S11, ESI†). Their accession number AXN72980, Fig. S1, ESI†) converted GGPP into
conversion with DdTPS9 gave stereoselectively labelled isoto- nephthenol (6) and minor amounts of cembrene A (Fig. S26,
pomers of (À)-4 with known configurations at the deuterated ESI†). The main product (S)-(+)-6 was isolated and identified by
carbons that allowed to infer the configurations of the native NMR spectroscopy (Table S4 and Fig. S27–S33, ESI†) and from its
stereogenic centres of (À)-4 as shown in Fig. 1.
optical rotation, [a]D20 = +45.5 (c = 0.67, C6D6). The absolute
The proposed cyclisation mechanism to 4 starts from FPP by configuration was confirmed by the stereoselective deuteration
isomerisation to nerolidyl diphosphate (NPP), followed by strategy (Fig. S34 and S35, ESI†). The diterpene alcohol (+)-6 has
cyclisation to the bisabolyl cation (A+). A 1,5-proton shift to B+ previously been isolated from a gorgonian octocoral Eunicea sp.
and cyclisation to the cuprenyl cation (C+) is succeeded by from Columbia,36 while the enantiomer (À)-6 is the main
two 1,2-methyl migrations via D+ to E+. Cyclisation to F+ and product of a type I diterpene synthase from Streptomyces sp.
deprotonation result in 4. The feasibility of this mechanism, SANK 60404,37 but no enzyme for (+)-6 was known before.
especially regarding the 1,5-proton migration, was supported by
DFT calculations by Hong and Tantillo,27 but an experimental
study is lacking. Incubation of (R)- and (S)-NPP28 with DdTPS9
demonstrated the selective and efficient transformation of the
S enantiomer into 4, while (R)-NPP yielded a mixture of 4 and
(E)-b-farnesene (Fig. S12, ESI†). Because (E)-b-farnesene is not a
native product of DdTPS9, (S)-NPP is likely the intermediate along
the cyclisation cascade from FPP to 4. The 1,5-proton shift from A+
to B+ was investigated by incubation of (2-2H)DMAPP16 with IDI
followed by heat inactivation and addition of (2-13C)DMAPP,29
Scheme 2 Cyclisation mechanism from GGPP to 5 by DpTPS10.
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The catalytic activity of TPSs depends on several highly
Communication
conserved motifs, as demonstrated by site-directed mutagen-
esis in conjunction with crystallographic data.38 This includes
the aspartate-rich motif (DDxx(x)(D,E)) and the NSE triad
(ND(L,I,V)xSxxxE) that are involved in binding of the Mg2+
cofactor,39–41 the C-terminal RY pair42 and the pyrophosphate
sensor, a highly conserved Arg residue that forms hydrogen
bridges to the pyrophosphate moiety of the substrate.43 More
recently, a structural role for two highly conserved Leu (some-
times substituted by Ile, Val or Met) and Pro residues located 14
and 21 positions upstream of the aspartate-rich motif at the
link between helices C and D, was supported by site-directed
mutagenesis that resulted in significantly reduced catalytic
activity and sometimes in poor enzyme yields.26,44,45 Further
experiments demonstrated that a salt bridge between a highly
conserved Arg on helix F and a Glu on helix G are important for
efficient catalysis.26,44,45 An alignment of several characterised
bacterial and amoebal TPSs with the three TPSs from Dictyos-
telium investigated in this study shows that the highly con-
served residues that are generally found in bacterial and fungal
TPSs are also present in TPSs from amoebae (Fig. S36, ESI†).
Only the salt bridge between R255 and E313 (positions accord-
ing to the alignment) is not observed in the enzymes reported
here. A general feature of all the aligned TPSs is a highly
conserved Asn located eight or nine residues downstream of
the NSE triad (N364). This residue is in the second active site
Scheme 3 Bromonium ion induced terpene cyclisations.
sphere and forms a hydrogen bridge to the Glu residue of the of 5 with NBS yielded four products (for NMR data cf. Tables S5–S8
NSE triad, as is evident from the crystal structure of selina- and Fig. S40–S70, ESI†), two of which represent the bromonium
4(15),7(11)-diene synthase (SdS, Fig. S37, ESI†),43 but has never ion induced cyclisation products 12 and 13. Unexpectedly, the
been tested by site-directed mutagenesis for any TPS. To exocyclic C14QC15 double bond was isomerised to an endocyclic
investigate the importance of this residue for catalytic activity, C10QC14 olefine in these materials. Furthermore, two products
the enzyme variants N236A and N236D of DdTPS9, N249A and 14 and 15 with an additional double bond were isolated that
N249D of DpTPS10, and N243A and N243D of DpTPS11 were require an oxidation step besides the bromonium ion induced
constructed by PCR-based site-directed mutagenesis. While cyclisation–isomerisation reactions.
most of these enzyme variants turned out to be insoluble
In summary, three TPSs from the social amoebae D. discoideum
(Fig. S1, ESI†), pointing to an important structural function of and D. purpureum were investigated and their products were
the conserved Asn, the N236A variant of DdTPS9 was obtained identified as (À)-b-barbatene (DdTPS9), (À)-b-araneosene (DpTPS10)
as a soluble protein, that exhibited a strongly reduced activity in and (S)-(+)-nephthenol (DpTPS11). While no barbatene synthase and
comparison to the wildtype enzyme (Fig. S38, ESI†). Taken no b-araneosene synthase was known so far, DpTPS11 yielded the
together, these results underpin the importance of this newly opposite enantiomer as a previously described enzyme from
discovered highly conserved residue for type I TPSs.
Streptomyces, demonstrating that social amoebae are an interest-
Vanadium-dependent bromoperoxidases (V-BrPOs) can catalyse ing source for the discovery of enzymes with new functions.52 The
bromonium ion induced polyene cyclisations of terpenes, leading to enzyme mechanisms were investigated through isotopic labelling
interesting structural diversifications as exemplified by the conver- experiments and by site-directed mutagenesis of a highly con-
sion of (R)-nerolidol (7) into a-snyderol (8, Scheme 3).46,47 A biomi- served Asn directly downstream of the NSE triad that is important
metic bromonium ion induced cyclisation was first described by van for catalytic activity. A sequence alignment with several charac-
Tamelen and Hessler using N-bromosuccinimide (NBS),48 but with terised TPSs from bacteria and fungi revealed that this Asn
open chain molecules such as methyl farnesenoate the conversion residue is also conserved in these enzymes, suggesting that this
into cyclic compounds is inefficient. For this purpose, more specia- residue may be of general importance for the catalytic activity of
lised reagents such as Et2SBrÁSbCl5Br were developed.49 More TPSs also from these kingdoms. Because of the accessibility of
efficient conversions have been reported with NBS for strained chloride and bromide in sea water, halogenated natural products
medium ring terpenes such as humulene (9) that results in two were frequently reported from marine organisms,53,54 and their
products 10 and 11 in ca. 20% yield each.50 A similar ring system is chemical synthesis often requires special methodology.55 The
present in 5, a compound for which the chemistry is poorly conversion of 5 into the brominated compounds 12 and 13 using
explored with just a few reported oxidative transformations,32,51 NBS described here resembles reactions that could potentially
but no halogenated derivatives have been prepared. Treatment also be catalysed by V-BrPOs. Since 5 is known from the marine
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Conflicts of interest
There are no conflicts to declare.
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