Two Diterpene Synthases for Spiroalbatene and Cembrene A from Allokutzneria albata

Article abstract of DOI:10.1002/anie.201800385

Two bacterial diterpene synthases from the actinomycete Allokutzneria albata were investigated, resulting in the identification of the structurally unprecedented compound spiroalbatene from the first and cembrene A from the second enzyme. Both enzymes wer

Full text of DOI:10.1002/anie.201800385

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Accepted Article
Title: Two Diterpene Synthases for Spiroalbatene and Cembrene A
from Allokutzneria albata
Authors: Jan Rinkel, Lukas Lauterbach, Patrick Rabe, and Jeroen
Sidney Dickschat
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201800385
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10.1002/anie.201800385
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Two Diterpene Synthases for Spiroalbatene and Cembrene A
from Allokutzneria albata
Jan Rinkel,[a] Lukas Lauterbach,[a] Patrick Rabe[a] and Jeroen S. Dickschat*[a]
Abstract: Two bacterial diterpene synthases from the actinomycete
Allokutzneria albata were investigated, resulting in the identification of
the structurally unprecedented compound spiroalbatene from the first
and cembrene A from the second enzyme. Both enzymes were deeply
investigated for their mechanisms by isotopic labelling experiments,
site-directed mutagenesis, and variation of metal cofactors and pH.
For spiroalbatene synthase the pH- and Mn2+-dependent formation of
the byproduct thunbergol was observed that is biosynthetically linked
to spiroalbatene.
purification (Figure S2, accession numbers are given in the SI).
The first enzyme did not accept GPP, FPP and GFPP, but GGPP
was efficiently converted into a diterpene hydrocarbon (Figure S3).
The compound was purified and its structure was elucidated by
NMR spectroscopy (Table S2, Figures S4-S10). The most
relevant 1H,1H-COSY, HMBC and NOESY correlations are shown
in Scheme 1A, resulting in the structure of a spirotetracyclic
diterpene with novel skeleton for which we propose the name
spiroalbatene (1). Thus, the DTS was identified as spiroalbatene
synthase (SaS).
The astonishing structural complexity of terpenes is reflected by
hundreds of known polycyclic carbon skeletons. These are all
formed by terpene synthases (TS) in a single enzymatic step from
a few linear oligoprenyl diphosphate (OPP) precursors, including
geranyl diphosphate (GPP) for monoterpenes (C10), farnesyl PP
(FPP) for sesquiterpenes (C15), geranylgeranyl PP (GGPP) for
diterpenes (C20), and geranylfarnesyl PP (GFPP) for
sesterterpenes (C25).[1] Starting from the pentalenene synthase
from Streptomyces exfoliatus,[2] previous research has resulted in
the discovery of several bacterial mono- (MTSs) and
sesquiterpene synthases (STS).[1] In contrast, only a few type I
diterpene synthases (DTSs) including those for cyclooctat-9-en-
7-ol (CotB2), spiroviolene, tsukubadiene, hydropyrene, 18-
hydroxydolabella-3,7-diene, and spata-13,17-diene have been
characterised,[3] with spata-13,17-diene synthase as the first
bacterial enzyme also making sesterterpenes.[3f] Because GGPP
has more options to react in comparison to GPP and FPP, the
chance to discover new compounds is higher for diterpenes than
for mono- and sesquiterpenes. It is not possible to predict directly
from the amino acid sequence of a TS which substrate preference
it has, but a phylogeny of TSs can unravel candidates with a
relationship close enough to a characterised DTSs to suspect
DTS activity, and distant enough to ensure that the TS does not
make the same product as the known enzyme. Using this
approach, we have identified two TSs from the tropical soil isolate
Allokutzneria albata DSM 44149[4] with close relationship to other
bacterial DTSs, as can be seen from a phylogenetic tree
constructed from 2728 bacterial TSs (Figure S1).
Scheme 1. Spiroalbatene (1). A) The carbon numbering for 1 indicates which
carbon derives from which carbon of GGPP by same number. Bold lines: 1H,1H-
COSY correlations, single-headed arrows: HMBC correlations, double-headed
arrows: NOESY correlations. B) Enzymatic synthesis of (13C20)-1. Grey dots
represent 13C atoms and bold lines 13C,13C-COSY correlations.
Further support for the structure of 1 was obtained by 13C,13C-
COSY NMR. For this purpose, we prepared completely
isotopically substituted (13C20)GGPP from (13C15)FPP.[6] During
our previous synthesis of this compound also (13C5)-3,3-
dimethylallyl alcohol was made available that was converted into
(13C5)DMAPP. Incubation of both compounds with the isopentenyl
diphosphate isomerase (IDI) from Serratia plymuthica, the GGPP
synthase (GGPPS) from Streptomyces cyaneofuscatus,[3c] and
SaS resulted in the efficient production of (13C20)-1 (Scheme 1B).
Analysis of the compound by 13C,13C-COSY NMR (Figure S11)
resulted in the observation of crosspeaks for all C-C-
connectivities apart from one.
The genes for both enzymes were cloned into the expression
vector pYE-Express[5] by homologous recombination in yeast,
followed by gene expression in Escherichia coli and protein
[a]
Prof. Dr. Jeroen S. Dickschat, Jan Rinkel, Lukas Lauterbach, Dr.
Patrick Rabe
Kekulé-Institute of Organic Chemistry and Biochemistry
University of Bonn
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany
E-mail: dickschat@uni-bonn.de
A biosynthetic model for 1 is shown in Scheme 2. Starting from
GGPP, a 1,14-cyclisation to cation A is followed by a 1,2-hydride
migration to B. Another 1,2-hydride shift to C and ring closure
results in D that upon a third 1,2-hydride shift and cyclisation
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201800385
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yields the cyclopropane intermediate E. Opening of the three-
membered ring with concomitant ring closure produces F. If the
E-to-F conversion is concerted, a secondary cation intermediate
can be avoided. A last ring closure and deprotonation result in 1.
This biosynthetic hypothesis was tested by conversion of all
twenty isotopomers of (13C)GGPP that were prepared by
synthesis or enzymatically from corresponding 13C-labelled FPP
and IPP isotopomers.[3c,6] For all substrates the label ended up in
a position as expected for the model (Figure S12).
configuration at C1.[11] After conversion of the enantioselectively
deuterated substrates with GGPPS and SaS the defined
stereochemical anchors at the deuterated carbons were used to
solve the absolute configuration of 1 by determining the relative
configurations of the stereospecifically deuterated products. The
additional 13C substitution was introduced for highly sensitive
product analysis by HSQC (Figures S16 and S17). The results
with all four probes consistently pointed to the absolute
configuration of 1 as shown in Scheme 1.
The elementary steps involving hydrogen migrations or
deprotonations were investigated by use of (stereo)selectively
deuterated probes. In several cases an additional 13C substitution
was introduced to enhance the carbon’s signal in 13C-NMR
analyses and to make resulting 13C-2H bonds observable by triplet
signals. The first 1,2-hydride shift from A to B was followed by
conversion of (14-2H)GGPP with SaS, prepared from IPP and (2-
2H)DMAPP[3e] with Streptomyces coelicolor FPP synthase
(FPPS)[7] and GGPPS.[3c] GC/MS analysis of the product showed
incorporation of one deuterium by an increased molecular ion,
while the fragment ion of 1 at m/z = 229 representing the loss of
the iPr group did not change (Figure S13A). Therefore, deuterium
incorporation was located in the iPr group as expected. A 1,3-
hydride migration from C1 into the iPr group as an alternative to
the two sequential 1,2-hydride migrations from A to C was ruled
out by incubation of (S)- and (R)-(1-2H)GGPP[3c] with SaS that
yielded products with a fragment ion at m/z = 230, meaning that
deuterium stayed within the tetracyclic core structure of 1 in both
cases (Figure S13B). The 13C-labelling from (16-13C)- and (17-
13C)GGPP showed a scrambling (Figure S12) that was previously
observed for other terpene cyclisations with 1,2-hydride
migrations into the iPr group[3e,8] and reflects the similar distances
for the migrating hydrogen to the two diastereotopic faces of the
cation in A. In contrast, for 1,3-hydride migrations always a strict
stereochemical course is observed.[9] The stereochemical course
for the second 1,2-hydride shift from B to C was investigated by
conversion of (S)- and (R)-(1-13C,1-2H)GGPP[3e] with SaS. While
the S-enantiomer yielded a product with a singlet for C1 in the
13C-NMR, the R-enantiomer resulted in a product with a triplet,
establishing the specific migration of the pro-S-hydrogen from C1
of GGPP (Figure S14A). The 1,2-hydride transfer from D to E was
investigated with (3-13C,2-2H)GPP, synthesised according to a
published route (Scheme S1),[3c] that was elongated with IPP and
GGPPS to (11-13C,10-2H)GGPP followed by cyclisation with SaS.
The product showed the expected triplet in the 13C-NMR spectrum
(Figure S14B). The terminal deprotonation step to 1 proceeds
from C4 of GGPP. Which of the two enantiotopic protons is lost
can be addressed by stereoselective deuteration. For this
purpose, (E)- and (Z)-(4-2H)IPP were synthesised (Scheme S2)
and used for the GGPPS-catalysed elongation of FPP to obtain
(S)- and (R)-(4-2H)GGPP (Figure S15). The stereochemical
course for this reaction includes attack of IPP at C4 from the Si
face.[10] Further conversion with SaS revealed specific loss of
deuterium from (E)-(4-2H)IPP, but retainment from (Z)-(4-2H)IPP,
revealing a deprotonation of G as shown in Scheme 2.
Scheme 2. Cyclisation mechanism from GGPP to 1 by SaS, hypothetical
formation of ent-2 from A, and formation of 3 from C.
The second enzyme from A. albata also did not accept GPP, FPP
and GFPP, but efficiently converted GGPP into one main and a
few side products (Figure S18). The main product was purified
and showed identical NMR data to those of cembrene A (Table
S3),[12] while the optical rotation was []D20 = +12.0 (c 0.13, CHCl3).
With the published optical rotation for the synthetic S ([]D
=
+19.5)[13] and R enantiomers ([]D = –12, c 0.85, CHCl3),[12]
these data pointed to the structure of (S)-(+)-cembrene A (2,
Scheme 3) and identified the enzyme from A. albata as cembrene
A synthase (CAS). The cyclisation mechanism from GGPP to 2
requires a 1,14-cyclisation to the cembranyl cation (ent-A) and
deprotonation from one of the geminal Me groups. SaS could
potentially yield ent-2 from intermediate A, reflecting the
phylogenetic distance between the two enzymes, but ent-A is not
observed from SaS. Incubation of (16-13C)- and (17-13C)GGPP
20
We have recently developed
a method that uses the
stereoselectively deuterated precursors (S)- and (R)-(1-13C,1-
2H)FPP and (S)- and (R)-(1-13C,1-2H)GPP for determination of the
absolute configurations of terpenes.[3c] The elongation of OPPs
with IPP by OPP synthases proceeds with inversion of
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revealed a distribution of labelling and therefore a relaxed
stereochemical course for the deprotonation step (Figure S19).
Incubation of (S)- and (R)-(1-13C,1-2H)GGPP with CAS and
product analysis by HSQC showed inversion of configuration at
C1 for the 1,14-cyclisation of GGPP (Figure S20).
residues are placed at the link between helices C and D (Figure
S24) and are likely of structural relevance. In CAS the
corresponding position to Pro61 is naturally occupied by Ala. In
this case the A64P variant has lower activity than the wildtype, but
only with Mg2+, while no effect was observed with Mn2+.
A highly conserved Glu residue seen in >93% of bacterial TSs is
occuppied by Gln160 in SaS. In SdS this residue is involved in
metal cofactor binding (Figure S25). While gene expression of the
SaS Q160E variant worked well, the enzyme showed no activity
regardless of using Mg2+ or Mn2+. For CAS the opposite exchange
(E179Q) gave a strongly reduced activity with both metal cations,
and the E179K variant was inactive. An interesting structural
feature of SdS is a salt bridge between a highly conserved Arg144
and a less conserved Glu192. We have recently shown for SpS
that the D217E mutation within the salt bridge (Arg169 and
Asp217) results in an inactive enzyme variant in combination with
Mn2+, while an increased activity was observed with Mg2+.[3f] The
corresponding residues for SaS (Arg145 and Glu193) were
mutated with shortening of the salt bridge by the R145K or E193D
exchanges, resulting in a reduced (with Mg2+) or complete loss of
activity (with Mn2+). For the R145M variant an only slightly
decreased activity was observed for both metal cofactors. Of
particular interest is the observation that both SpS[3f] and CAS
have a naturally short salt bridge with participation of an Asp
instead of the more regular Glu, and both enzymes are more
efficient with Mn2+ than with Mg2+. For CAS the D212E mutation
resulted in a strongly decreased activity with Mg2+ and Mn2+.
These data suggest an influence of the length of this salt bridge
likely by alteration of the angle of slope between helices G and F
on the active site architecture, with consequences for metal
binding. A short salt bridge with participation of Asp seems to
favour catalysis with Mn2+, while a long salt bridge with Glu fosters
Mg2+ binding, but further experiments with other TSs are required
to investigate whether this finding is generalisable.
Scheme 3. Cyclisation mechanism from GGPP to 2.
Type I TSs generally exhibit the same -helical fold as first
recognised for avian FPPS.[14] Bacterial enzymes, apart from
geosmin synthase with two  domains,[15] adopt a single  domain
architecture with several highly conserved motifs involved in
binding of the metal cofactor and substrate recognition.[16] This
includes the aspartate-rich motif DDXX(X)(D,E), the NSE triad
ND(L,I,V)XSXX(R,K)(E,D), the diphosphate (PP) sensor (R near
the helix G break), and the RY dimer near the C-terminus. For
SaS the Mg2+ cofactor can be substituted by Mn2+ resulting in a
reduced activity of 27±17%, while for CAS a higher activity
(172±12%) is observed with Mn2+ (Figure S21). No activity was
observed with GGPP and SaS, if no metal cofactor was added,
demonstrating that not residual Mg2+ in buffers or enzyme
preparations accounts for activity in the experiments with Mn2+
(and vice versa).
The importance of several conserved residues in TSs for catalytic
activity has been demonstrated by site-directed mutagenesis
(SDM, critical residues identified in previous mutational studies
are shown in bold above).[3f,15,17] SaS exhibits all the conserved
motifs as usual, with the exception of a Gly instead of the Ser in
the NSE triad (Figure S22 and S23). Among all >50 characterised
bacterial TSs and their nearly 2500 close homologs from
sequenced bacteria (Figure S1), in only four cases a substitution
of this Ser is observed (Table S4). Installation of Ser by SDM
(G229S) gave no soluble enzyme, suggesting that Gly with its
conformational flexibility is of structural importance for enzyme
folding of wildtype SaS. The crystal structure of selina-
4(15),7(11)-diene synthase (SdS)[17d] shows hydrogen bridges
between the PP sensor Arg178, a highly conserved Tyr174 found
in >90% of bacterial TSs, and the first Asp225 of the NSE triad
(Figure S23). In SaS and in ca. 5% of bacterial TSs a Phe175 is
found instead of Tyr. The F175Y variant gave a good enzyme
yield, but the activity dropped to 28±6% with Mg2+, and a similar
loss of activity was observed with Mn2+.
A highly conserved Pro was identified 22 residues downstream of
the PP sensor. In SdS this residue is located at the bottom of helix
H and seems to have an important structural function (Figure S26).
In a few TSs the corresponding position is occupied by Thr or Ser,
but the P201T variant of SaS showed a significantly reduced
activity with both metal cofactors.
SaS was also investigated for an effect of changing the pH from
pH = 7.4 used in the experiments above to pH = 8.2. While no
significant change in the product profile was observed with the
wildtype enzyme in combination with Mg2+, incubations with Mn2+
resulted in a new byproduct (Figure S3). The compound was
isolated and identified by NMR (Table S5 and Figures S27-S33)
as thunbergol (3). Its absolute configuration of (1R,4S)-3 was
20
evident from the optical rotation ([]D = –48, c 0.85, CHCl3) by
20
comparison to (1S,4R)-3 from Pseudotsuga menziesii ([]D
=
+75.2, c 1.2, CHCl3),[18] establishing another example for a
bacterial TS producing the opposite enantiomer as in plants.[1,19]
Compound 3 can be formed via the same intermediate C as 1
(Scheme 2) which is corroborated by the same configuration of
the stereocentre at C14 for both products. The formation of 1 and
3 via C as a common intermediate is also mechanistically
supported: Incubation of (2-2H)DMAPP with IPP, GGPPS and
SaS followed by GC/MS analysis of the product 3 revealed the
same 1,2-hydride shift from A to B as determined for 1 (Figure
S34), again with a distribution of labelling from C16 and C17
For spata-13,17-diene synthase (SpS) from Streptomyces
xinghaiensis a critical role of the highly conserved Pro and Leu
located 21 and 14 positions upstream of the Asp-rich motif was
recently demonstrated.[3f] The corresponding L72A variant of SaS
gave no soluble protein, while P65A yielded wildtype levels of
enzyme, but the activity dropped significantly with Mg2+ and Mn2+.
As can be seen in the crystal structure of SdS,[17d] these two
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E. Dolja, T. Schmitz, B. Nubbemeyer, T. H. Luu, J. S. Dickschat, Angew.
Chem. Int. Ed. 2017, 56, 2776; d) J. S. Dickschat, J. Rinkel, P. Rabe, A.
Beyraghdar Kashkooli, H. J. Bouwmeester, Beilstein J. Org. Chem. 2017,
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(Figure S35). And as observed for 1, the enzymatic conversion of
(R)- and (S)-(1-13C,1-2H)GGPP with SaS gave evidence for a
stereoselective migration of the 1-pro-S hydrogen to C also for 3
(Figure S36).
None of the diterpenes identified from SaS or CAS were observed
in headspace extracts from A. albata, while spiroviolene was
reported in our previous study,[3c] suggesting that both enzymes
are not expressed in laboratory cultures. Alternatively, 1 may be
oxidised to an unknown product by a genetically clustered
cytochrome P450 (Figure S37 and Table S6).
In summary, we have identified the products of two DTSs from A.
albata, one of them exhibiting the unprecedented structure of
spiroalbatene. While the general principles of terpene
biosynthesis are well understood, the recently accumulating data
from isotopic labelling experiments, enzyme crystallisations,
enzyme mutagenesis and variation of incubation conditions lead
to more detailed insights that may allow for predictive approaches
how to get access to new terpenes in the future. The phylogenetic
tree of bacterial TSs (Figure S1) shows that the products of many
enzymes are known today, but there are also many dark areas for
continuing research on this interesting enzyme class.
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This work was funded by the DFG (DI1536/7-1) and by the Fonds
der Chemischen Industrie with a PhD sholarship to JR. We thank
Paolina Garbeva (Wageningen) for Serratia plymuthica PRI-2C.
Keywords: biosynthesis • enzyme mechanisms • isotopes • soil
microorganisms • terpenes
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COMMUNICATION
COMMUNICATION
Two diterpene synthases from
Allokutzneria albata were investigated
for their products, resulting in the
identification of the structurally
complex compound spiroalbatene and
of cembrene A. The enzyme
mechanisms were studied in isotopic
labelling experiments, by mutations
and variations of incubation
Jan Rinkel, Lukas Lauterbach, Patrick
Rabe and Jeroen S. Dickschat*
Page No. – Page No.
Two Diterpene Synthases for
Spiroalbatene and Cembrene A from
Allokutzneria albata
conditions. For spiroalbatene
synthase changed incubation
conditions resulted in the formation of
thunbergol.
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