Spata-13,17-diene Synthase—An Enzyme with Sesqui-, Di-, and Sesterterpene Synthase Activity from Streptomyces xinghaiensis

Article abstract of DOI:10.1002/anie.201711142

A terpene synthase from the marine bacterium Streptomyces xinghaiensis has been characterised, including a full structure elucidation of its products from various substrates and an in-depth investigation of the enzyme mechanism by isotope labelling experiments, metal cofactor variations, and mutation experiments. The results revealed an interesting dependency of Mn²⁺ catalysis on the presence of Asp-217, a residue that is occupied by a highly conserved Glu in most other bacterial terpene synthases.

Full text of DOI:10.1002/anie.201711142

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Accepted Article
Title: Spata-13,17-diene Synthase, an Enzyme with Sesqui-, Di- and
Sesterterpene Synthase Activity from Streptomyces xinghaiensis
Authors: Jan Rinkel, Lukas Lauterbach, and Jeroen Sidney Dickschat
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711142
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Spata-13,17-diene Synthase, an Enzyme with Sesqui-, Di- and
Sesterterpene Synthase Activity from Streptomyces xinghaiensis
Jan Rinkel,[a] Lukas Lauterbach[a] and Jeroen S. Dickschat*[a]
This work is dedicated to Prof. Dr. Stefan Schulz on the occasion of his 60th birthday.
Abstract:
A
terpene synthase from the marine bacterium
full
(PP) sensor Arg-173, the NSE triad (119NDWYSLGKE), and the
307RY dimer (Figure S1). The closest characterised homolog of
this enzyme is the (+)-epi-cubenol synthase from Streptomyces
griseus NBRC 13350 with 51% identical amino acid residues.[8]
The protein was purified (Figure S2) and incubated with GPP,
FPP, GGPP and GFPP. Among these substrates GPP was not
accepted, while GGPP and GFPP were converted efficiently into
a di- or sesterterpene hydrocarbon, respectively, accompanied by
a few side products (Figure 1). The diterpene hydrocarbons 1 and
2 were also detected in headspace extracts from S. xinghaiensis
(Figure S3).
Streptomyces xinghaiensis was characterised, including
a
structure elucidation of its products from various substrates and an in-
depth investigation of the enzyme mechanism by isotopic labelling
experiments, metal cofactor variations, and mutation experiments.
The results revealed an interesting dependency of Mn2+ catalysis on
the presence of Asp-217, a residue that is occupied by a highly
conserved Glu in most other bacterial terpene synthases.
In terms of their structural variability, complexity, and distribution
terpenes constitute one of the most successful classes of natural
products. They are synthesised from geranyl (GPP), farnesyl
(FPP), geranylgeranyl (GGPP), geranylfarnesyl diphosphate
(GFPP), and even larger isoprenoid oligomers by terpene
synthases (TS) that generate
a usually polycyclic carbon
framework with multiple stereogenic centres in just one enzymatic
step. Although multi-product TSs such as MtTPS5 from Medicago
truncatula are known,[1] many enzymes selectively produce a
single compound with astonishing accuracy. This precision is
particularly remarkable, because the action of a TS on a substrate
seems to be limited to its ionisation, either by abstraction of
diphosphate (type I enzymes) or by protonation (type II), and to
provide a shaped and essentially water-free cavity to arrange the
substrate in a reactive conformation. The reaction cascade via
cationic intermediates makes then use of the inherent substrate
reactivity.[2] While several type I mono- and sesquiterpene
synthases (STS) have been reported from bacteria,[3] the only
characterised diterpene synthases (DTSs) of this class for which
the intriguing product structures and enzyme mechanisms have
been thoroughly studied are the enzymes for cyclooctat-9-en-7-ol,
spiroviolene, tsukubadiene, and 18-hydroxydolabella-3,7-diene,
and the multi-product DTS for hydropyrene.[4] Here we present the
characterisation of a TS from Streptomyces xinghaiensis, a
marine actinomycete that has been isolated from sediments near
Dalian, China,[5] that shows a broad substrate spectrum and
interesting metal cofactor dependency.
Figure 1. Total ion chromatograms of products obtained from an incubation of
A) GGPP and B) GFPP with SpS. Arrows point to minor enzyme products,
numbers at peaks refer to compound numbers in Scheme 1.
The main diterpene 1 was isolated by column chromatography,
yielding 6 mg (14%) of pure 1, and its structure was elucidated by
NMR spectroscopy as spata-13,17-diene (Table S2, Figures S4–
S10, most relevant 2D-NMR correlations are highlighted in
Scheme 1), establishing the TS from S. xinghaiensis as spata-
13,17-diene synthase (SpS). Two minor products could also be
isolated and were identified by NMR as prenylkelsoene (2, 0.6 mg,
1%) and the known compound cneorubin Y[9] (3, 0.8 mg, 2%)
(Tables S3 and S4, Figures S11–S23). The latter is not detectable
by GC/MS, likely because it contains a Cope system resulting in
thermal instability as described for germacrenes.[10] The
diterpenes 1 and 2 are new natural products.
GFPP was also accepted by SpS, resulting in the formation of a
main and a few side products. The main product (0.9 mg, 1%)
was identified by NMR (Table S5, Figures S25–S31) and GC/MS
(Figure S32) as the higher homolog of 1, prenylspata-13,17-diene
(6). The homolog of 2, geranylkelsoene (7), was also tentatively
identified by GC/MS based on the characteristic loss of ethylene
from the cyclobutane portion (Figure S33). The sesterterpenes 6
and 7 are new natural products, and SpS is the first bacterial TS
reported to have sesterterpene synthase activity.
The gene for an unknown TS from S. xinghaiensis S187
(accession no. WP_095757924) was cloned into the expression
vector pYE-Express[6] by homologous recombination in yeast and
expressed in Escherichia coli. The predicted gene product
exhibited all highly conserved motifs for a functional type I TS,[7]
including the aspartate-rich motif (74DDQLD), the pyrophosphate
[a]
Prof. Dr. Jeroen S. Dickschat, Jan Rinkel, Lukas Lauterbach
Kekulé-Institute of Organic Chemistry and Biochemistry
University of Bonn
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany
E-mail: dickschat@uni-bonn.de
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201711142
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The corresponding sesquiterpene to
3
is the widespread
of 6/7 and 8/9, respectively, via the hypothetical intermediates 3a
and 3b. In contrast, the enzymatic conversion of FPP stopped at
11 and did not proceed to tricyclic analogs of 1 and 2, but
produced major amounts of 12 by alternative deprotonation of A,
and 13 by 1,3-hydride migration and deprotonation.
compound bicyclogermacrene (11). Notably, FPP was also
accepted by SpS and converted into a mixture of 11, germacrene
A (12) and germacrene D (13) that were partially detected as their
Cope rearrangement products by GC/MS (Figure S34, Table S6).
Compound 12 was also isolated and characterised by NMR,
showing identical data to those previously reported.[11]
Scheme 1. Products of SpS and structures of related natural products. The
carbon numberings for 1 – 3 follow the GGPP numbering to indicate the
biosynthetic origin of each carbon. This numbering is different to previously
introduced numbering systems (cf. comment on page 1 of SI).
Scheme 2. Biosynthetic mechanism for SpS.
Poduran (10) is a tetraterpenoid with the same tricyclic core as in
2 and a saturated side chain that was reported from the springtail
Podura aquatica.[12] The broad substrate specificity of SpS
suggested that a similar enzyme may be responsible for the
biosynthesis of 10. This prompted us to investigate whether
14,15-dihydro-GGPP can also be converted. For this purpose,
10,11-dihydro-FPP was synthesised (Scheme S1), elongated
with IPP by the GGPP synthase (GGPPS) from Streptomyces
cyaneofuscatus,[4b] and converted by SpS, yielding 1.8 mg (5%)
of spat-13-ene (8) (Table S7, Figures S32 and S35–S41). A minor
product was tentatively identified from its mass spectrum as
isopentylkelsoene (9) that showed a loss of 28 Da from the
molecular ion (Figure S33), as reported for 10.[12]
The biosynthesis of 1 and 2 can be rationalised by 1,10-
cyclisation to A, followed by deprotonation with formation of a
cyclopropane ring to 3. This neutral intermediate can be
reprotonated at C-3 for a second cyclisation to B. This cation can
react by two alternative ring openings of the cyclopropane, either
via pathway a) to C that is the precursor of 1, or via pathway b) to
D, the direct precursor of 2. Starting from GFPP or 14,15-dihydro-
GGPP, essentially the same reactions can explain the formation
The proposed biosynthetic pathway of Scheme
2 was
investigated by incubation of all twenty isotopologs of (13C)GGPP,
obtained by chemical synthesis or enzymatically from the
corresponding labelled FPP or IPP isotopologs using S.
cyaneofuscatus GGPPS,[4b,13] resulting in the incorporation of
labelling into the expected positions of 1–3 in all cases (Figures
S42–S44). In particular, these results demonstrate the formation
of 1 by cyclopropane ring closure to 3 and reverse ring opening
with carbon backbone rearrangement.
The sesquiterpene prespatane (bourbon-11-ene, 4) has been
reported from the sponge Cymbastella hooperi[14] and from
several liverworts[15] and actinobacteria.[16] In all these organisms
4 co-occurs with kelsoene (tritomarene, 5), but the different
relative orientation of the isopropenyl group seems to exclude a
simple biosynthetic mechanism via a common intermediate 11 as
we have found for the pairs 1+2 and 6+7. A comparison of the
13C-NMR data of 1 and 2 to those reported for 4 and 5[14] suggests
that the structure of 4 needs correction to 8-epi-4 that also better
fits into the biosynthesis scheme (Figure S45). The structural
revision for prespatane was recently also independently
described by Weng and coworkers.[17] In this report a biosynthesis
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of 8-epi-4 via germacrene A, B or C was suggested, but the larger
homolog 3 of bicyclogermacrene (11) as intermediate can better
explain the common formation of the two products 1 and 2.
The reprotonation of 3 at C-3 for the second cyclisation to B
(Scheme 2) was investigated by incubation of (3-13C)IPP[4b] and
FPP with GGPPS and SpS in D2O. The obtained product (3-13C,3-
2H)-1 showed a triplet in the 13C-NMR in agreement with
reprotonation at C-3 (Figure S46). The stereochemical course of
the deprotonation from A to 3 was investigated by enzymatic
conversion of (S)- and (R)-(1-13C,1-2H)GGPP[18] with SpS.
Labelled 1 obtained from (S)-(1-13C,1-2H)GGPP showed a singlet
different brown algae, including (13Z)-spata-13(15),17-diene (14)
and spatol (15, Scheme 3).[25] These compounds exhibit the
opposite absolute configuration as determined for 1.
in 13C-NMR, while (R)-(1-13C,1-2H)GGPP gave
a product
Scheme 3. Known spatanes from brown algae.
exhibiting a triplet due to 13C-2H spin coupling, establishing the
loss of the pro-S and retainment of the pro-R hydrogen from C-1
in the cyclisation to 3 which was also supported by GC/MS
analysis (Figure S47). Analogous results were obtained for 6 with
(S)- and (R)-(1-13C,1-2H)GFPP[13b] (Figure S48) and for 11 with
(S)- and (R)-(1-2H)FPP (Figure S49). For 13 a stereospecific shift
of the pro-S hydrogen into the isopropyl group was observed by
GC/MS (Figure S50). Assuming inversion of configuration at C-1
for the initial 1,10-cyclisation step to A as reported for other
terpene cyclisations,[19] these data are in favour of the absolute
configurations of the products of SpS as shown in Scheme 2. In
particular, the stereochemical course of the 1,3-hydride shift from
C-1 of FPP into the isopropyl group as for 13 was shown to be
indicative for the absolute configuration of sesquiterpenes.[20]
The absolute configuration of 12 was independently established
by compound isolation from its optical rotation ([]D20 = –7.2, c 0.5,
CCl4) and comparison to different literature data for 12 ([]D25 = –
Several TSs have been investigated by site-directed mutagenesis
(SDM), including the fungal trichodiene synthase and the bacterial
TSs for pentalenene, epi-isozizaene, (2Z,6E)-hedycaryol and
selina-4(15),7(11)-diene (Tables S8–S12).[7a,26] This work
underpinned the importance of highly conserved motifs such as
the Asp-rich motif,[7a,26c,f,g] the NSE triad,[7a] the RY dimer,[26a,b,g]
and the PP sensor[26g] for catalysis. A detailed analysis of 51
characterised bacterial type I TSs,[3] their close relatives with
presumably the same function from sequenced bacteria, and SpS
by sequence alignment resulted in the identification of four highly
conserved amino acid residues whose importance for catalysis
have not been shown so far. These include P83 and L90, located
21 and 14 positions upstream of the Asp-rich motif, and E184 and
E217 that are placed 19 positions upstream and 14 positions
downstream of the PP sensor (Table S13, SpS numbering, the
usually found E217 is altered to D217 in native SpS). P83A
exchange resulted in a poor yield of soluble enzyme and a
complete loss of activity (Figures S57 and S58). Also the L90A
variant showed a lower expression level and significantly reduced
activity (3%) compared to native SpS. The crystal structure of
selinadiene synthase from Streptomyces pristinaespiralis[26g]
shows that its corresponding residues (P61 and V68) have an
important structural role at the link between two -helices (Figure
S59). Their exchange in SpS may result in an incorrect enzyme
folding, causing poor activity and yield of enzyme. The efficiently
expressed E184Q variant of SpS was nearly inactive (0.5%). The
structure of selinadiene synthase shows that the corresponding
E159 is involved in Mg2+ binding (Figure S60) that may be critical
for activity. Exchange of D217 to the usual Glu (D217E) resulted
in an increased expression and catalytic efficiency (170%).
Comparison to the structure of selinadiene synthase reveals that
the corresponding E192 in helix G is part of a salt bridge to R144
in helix F (Figures S60 and S61). In SpS, D217 can substitute for
this function, but the D217E variation seems to fit better to the
structural requirements of a type I TS.
25
3.2, c 14.4, CCl4; []D = –26.8, c 1.0, CCl4) and its enantiomer
([]D25 = +42.1, c 1.0, CCl4).[21] Compound 12 was also converted
into its Cope rearrangement product -elemene in refluxing
20
toluene to yield a material of []D = +17.9 (c 0.06, CHCl3),
consistent with the earlier reported conversion of (+)-12 into (–)-
25
-elemene ([]D = –15.8 (c 0.50, CHCl3)).[20c] The absolute
configurations of 1 – 3 were investigated by conversion of
stereoselectively deuterated (R)- and (S)-(1-13C,1-2H)GPP and
(R)- and (S)-(1-13C,1-2H)FPP[4b] that were elongated to the
corresponding GGPPs using S. cyaneofuscatus GGPPS (Figures
S51–S56). This reaction is known to proceed with inversion of
configuration at C-1 of GPP and FPP.[22] The obtained
stereoselectively deuterated GGPP isotopologs were converted
into 1 – 3 by SpS. The installed stereochemical anchors with
known absolute configurations allowed to deduce the absolute
configurations at the other stereocentres simply by solving the
relative configurations of the obtained stereoselectively
deuterated products. The additional 13C label was introduced for
an efficient and sensitive product analysis by HSQC. The
deduced absolute configurations for 1 – 3 are in line with their
Catalysis by type I TSs requires a trinuclear cluster of divalent
cations (usually Mg2+ or Mn2+) that binds to the Asp-rich motif, the
NSE/DTE triad and the substrate’s PP to initiate its ionisation.[27]
Other divalent cations were sometimes reported to be ineffective
for catalysis,[28] but most of the recently described enzymes have
not been systematically investigated for their metal ion
dependency. Incubation experiments with SpS and various
cations (Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+) resulted in
efficient catalysis by Mg2+ and, with ca. threefold higher rates, by
Mn2+ (Figure S62), while all other cations gave no product.
biosynthetic relationship. The absolute configuration of
1
corresponds to that of prespatane (8-epi-4) from the alga
Laurencia pacifica.[17] The same absolute configuration as found
here for 3 can be assigned to 3 from Cneorum tricoccon based on
the same sign for optical rotations (found here: []D20 = –31, c 0.05,
acetone, reported: []D20 = –49.1, 0.3%, acetone).[23] The absolute
20
configuration of 2 ([]D = +20 (c 0.05, C6D6)) is the same as
reported for natural (+)-kelsoene that was established by
synthesis of its enantiomer.[24] The main product 1 of SpS is
structurally related to a series of spatanes that were reported from
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[11] A. M. Adio, C. Paul, H. Tesso, P. Kloth, W. A. König, Tetrahedron Asym.
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Interestingly, incubation of the highly productive D217E variant
with Mn2+ gave no diterpene product from GGPP, possibly
because this mutation causes a conformational rearrangement in
the helices F and G that disturbs the active site residues involved
in metal cofactor binding. A BLAST search and phylogenetic
analysis (Figure S63) revealed the presence of two closely related
homologs of SpS in Streptomyces albus NRRL F-4971
(WP_030543144, 89% identity) and in Streptomyces fradiae
ATCC 19609 (WP_050363727, 96%). Both enzymes also exhibit
an aspartate residue in the position corresponding to Asp-217 of
SpS. Future experiments will address whether the metal cofactor
requirement also for other bacterial TSs can be tuned by mutation
of this highly conserved Asp.
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This work was funded by the DFG (DI1536/7-1) and by the Fonds
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Keywords: biosynthesis • enzyme mechanisms • isotopes •
metal cofactors • terpenes
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10.1002/anie.201711142
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
A terpene synthase from
Jan Rinkel, Lukas Lauterbach and
Streptomyces xinghaiensis with
sesqui-, di, and sesterterpene
Jeroen S. Dickschat*
Page No. – Page No.
synthase activity was identified. The
enzyme mechanism was investigated
by isotopic labelling experiments. Site-
directed mutagenesis uncovered
several previously unrecognised
highly conserved residues that are
important for catalysis. One of these
residues was linked to Mg2+ versus
Mn2+ cofactor requirement.
Spata-13,17-diene Synthase, an
Enzyme with Sesqui-, Di- and
Sesterterpene Synthase Activity from
Streptomyces xinghaiensis
This article is protected by copyright. All rights reserved.