Mechanistic characterization of three sesquiterpene synthases from the termite-associated fungus: Termitomyces

Article abstract of DOI:10.1039/c8ob02744g

Three terpene synthases from the termite associated fungus Termitomyces were functionally characterized as (+)-intermedeol synthase, (-)-γ-cadinene synthase and (+)-germacrene D-4-ol synthase, with the germacrene D-4-ol synthase as the first reported enzyme that produces the (+)-enantiomer. The enzymatic mechanisms were thoroughly investigated by incubation with isotopically labeled precursors to follow the stereochemical courses of single reaction steps in catalysis. The role of putative active site residues was tested by site directed mutagenesis of a highly conserved tryptophan in all three enzymes and additional residues in (-)-γ-cadinene synthase that were identified by homology model analysis.

Full text of DOI:10.1039/c8ob02744g

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DOI: 10.1039/C8OB02744G
Organic and Biomolecular Chemistry
ARTICLE
Mechanistic Characterization of Three Sesquiterpene Synthases
from the Termite-Associated Fungus Termitomyces
a
b
b
a
Immo Burkhardt, Nina Kreuzenbeck, Christine Beemelmanns and Jeroen S. Dickschat*
Received 00th January 20xx,
Accepted 00th January 20xx
Three terpene synthases from the termite associated fungus Termitomyces were functionally characterized as (+)-
intermedeol synthase, (–)--cadinene synthase and (+)-germacrene D-4-ol synthase, with the germacrene D-4-ol synthase
as the first reported enzyme that produces the (+)-enantiomer. The enzymatic mechanisms were thoroughly investigated
by incubation with isotopically labeled precursors to follow the stereochemical courses of single reaction steps in catalysis.
The role of putative active site residues was tested by site directed mutagenesis of a highly conserved tryptophan in all three
enzymes and additional residues in (–)--cadinene synthase that were identified by homology model analysis.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Higher fungi are a prolific source of bioactive terpenoid natural
Introduction
products like the mycotoxins T2-toxin produced by Fusarium
sporotrichioides and PR-toxin found in Penicillium roqueforti,
5
6
With more than 80,000 members, terpenoids represent the
largest class of natural products. One important step towards
the structural complexity of terpenoids is the enzymatic
cyclization of strikingly simple oligoprenyl pyrophosphate
precursors by terpene synthases to produce (poly)cyclic
or the gibberellins, a group of phytohormones produced by
7
Fusarium fujikuroi. Most characterized fungal terpene
5
-7
synthases are encoded in the genomes of ascomycetes, which
comprise the largest number of known fungal species, while the
second-largest group, the basidiomycetes, remains a mostly
1
terpenes, often with several stereocenters, in a single reaction.
8
untapped source for these biosynthetic enzymes. To date the
These cyclizations commence by production of a carbocation
that is catalyzed by the terpene synthase. In the case of type I
terpene synthases, this is achieved by abstraction of
only characterized type I sesquiterpene synthases from
9
basidiomycetes originate from Omphalotus olearius, Stereum
1
0
11
12
hirsutum, Armillaria gallica and Coprinus cinereus, but
none of the enzymes have been investigated with respect to
their cyclization mechanisms. An especially interesting taxon of
basidiomycetes is the genus Termitomyces, whose members
live exclusively in close symbiosis with fungus growing termites
that actively cultivate the fungus in special chambers in their
1
pyrophosphate to yield an allylic cation. Subsequently, carbon-
carbon bonds are established in a cascade reaction by
nucleophilic attack of double bonds from the pre-folded
substrate in the enzyme’s active site, which determines the
regio- and stereochemical course of the reaction. Termination
takes place either by final deprotonation to produce terpene
hydrocarbons or by attack of a water molecule to yield terpene
1
3
nests as foodstock. The close interspecies relationship
requires recognition and interaction, that is most likely
mediated through volatile secondary metabolites such as
1
alcohols. Investigations of these complex reactions and the
interaction of the active site and the reacting substrate during
catalysis has made progress using protein crystallography,
1
4
terpenes. Indeed, a bioinformatical analysis of the draft
1
a,2
1
5
genome sequence of Termitomyces sp. J132, showed the
presence of 22 putative type I terpene synthase genes (STC1-
STC22). None of the genes could be affiliated with a putative
product, because no gene homologs had been characterized
before. In the presented study we chose three of the genes for
functional identification that encoded all conserved motifs,
which are needed for type I terpene synthase reactivity. Two of
the selected genes (STC4 and STC15) were expressed under
laboratory culture conditions and could be amplified from
mRNA using reverse transcriptase PCR. A third enzyme (STC9)
attracted our attention, because transcriptomic studies of
Termitomyces comb material and nodules showed high
3
4
isotopic labelling experiments and theoretical investigations,
but to reach a more comprehensive understanding of the
underlying principles of terpene cyclization reactions, more
enzymes and their detailed reaction mechanisms need to be
analyzed.
a. Kekulé-Institute of Organic Chemistry and Biochemistry, University of Bonn,
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany.
Email: dickschat@uni-bonn.de
b.
Leibnitz Institute for Natural Product Research and Infection Biology, Hans-Knöll-
Institute, Beutenbergstraße 11a, 07745 Jena, Germany.
Electronic Supplementary Information (ESI) available: Procedures for cloning,
enzyme expressions and purifications, in vitro experiments, product purifications expression levels, suggesting an ecological function of this
and site directed mutagenesis; analytical details of identified products; additional
sprectroscopical details of incubation experiments; amino acid sequences of
discussed enzymes; homology model analyses for the discussed cadinene synthases;
1
6
enzyme and its product. The sequence of this gene with
annotaded introns and exons is published in the NCBI database
NMR spectra. See DOI: 10.1039/x0xx00000x
under accession number KNZ74377 (locus tag J132_07087) and
This journal is © The Royal Society of Chemistry 20xx
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was obtained by gene synthesis. We report on the functional major enzymatic product, but -selinene (8) and V-iesweAlirnticelenOen(li9ne)
characterization of these genes via heterologous expression in were also detected (Figure S1). AnotherDmOiI:n1o0r.10co39m/Cp8oOuBn0d27w44aGs
Escherichia coli and purification of the corresponding enzymes, -elemene, which represents the thermal Cope rearrangement
a mechanistic investigation of their cyclization mechanisms by product of germacrene A (6) and is an artifact formed in the GC-
2
2
incubation with isotopically labeled precursors and probing of injector. A possible cyclization mechanism that leads to 1 and
different putative active site residues by site directed the minor compounds starts by abstraction of pyrophosphate
mutagenesis.
followed by a 1,10-cyclization to cation 5, which after
deprotonation yields germacrene (6) as neutral
A
a
intermediate (Scheme 1). Reprotonation of 6 and subsequent
ring closing gives rise to the cation 7. A final attack by water
Results and discussion
yields 1, while two alternative deprotonations of 7 can result in
either 8 or 9.
Putative terpene synthase genes in Termitomyces sp. J132 were
1
7
identified using antiSMASH 3.0. Candidate gene sequences for
STC4 and STC15 were obtained by primer-based amplification
of cDNA derived from RNA isolated from the close relative
Termitomyces sp. T153. Despite several attempts, a candidate
gene sequence for STC9 could not be retrieved from mRNA of
Termitomyces spp. and was therefore purchased via custom-
based gene synthesis. All three genes were cloned into the
8
13
1
4
4
1
-
OPP-
OPP 12
FPP
15
4
5
H+
-
H+
1
8
expression vector pYE-Express and heterologously expressed
in E. coli BL21. The enzymes were purified and incubated in vitro
with oligoprenyl diphosphate precursors. All three enzymes
only converted farnesyl pyrophosphate (FPP) into
sesquiterpenes or sesquiterpene alcohols, while there were no
reactions with geranyl pyrophosphate (GPP), geranylgeranyl
pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate
+
H O
2
H
H
HO
1
7
6
H+
-H+
-
(
GFPP). Incubations of the enzymes with FPP yielded a single
H
H
product from STC9 and mixtures of sesquiterpenes from STC4
and STC15. The (main) products were purified by column
chromatography in all three cases to yield a sesquiterpene
alcohol from STC4, a sesquiterpene hydrocarbon from STC9 and
a sesquiterpene alcohol from STC15 (for procedures, see
supporting information). Their structures were unambiguously
established by one and two-dimensional NMR analysis (Tables
S3 – S5) and measurement of their optical rotary powers. The
main product of STC4 was identified as (+)-intermedeol (1),
the single product of STC9 was (–)--cadinene (2) and the main
product of STC15 was (+)-germacrene D-4-ol (3) (Figure 1).
Bacterial enzymes, which produce 1, 2 and ent-3, but show only
low sequence homology to the here identified enzymes, were
characterized in our laboratories in previous studies with regard
8
9
Scheme 1: Cyclization mechanism for STC4 from FPP via 6 towards the main product 1
and the side products 8 and 9.
To follow the reprotonation of 6, an incubation experiment with
STC4 and (6- C)FPP in D
incubation mixture was extracted with deuterated benzene and
the extract was directly subjected to GC-MS and NMR analysis.
An enhanced mass by +2 Da of the molecular ion of 1 in the GC-
MS analysis confirmed uptake of one deuterium from D
the C-labeled compound (Figure S2). C-NMR analysis
showed a strongly enhanced triplet signal for C-1 due to H- C
J-coupling, indicating a deuterium directly bound to the labeled
13
2
O was conducted (Figure 2A). The
1
9
2
0
2
O to
2
1
13
13
2
13
1
carbon (Figure 2C). The stereochemical course of the
reprotonation was investigated by HSQC analysis, which
showed in comparison to the spectrum of unlabelled material
1
9,20,21
to their mechanisms.
To shed light on the stereochemical
details of the cyclization mechanisms leading to 1, 2 and 3 and
to allow a comparison with the already described enzymes, a
thorough investigation by incubation with different FPP
isotopomers was carried out for each enzyme.
(

Figure 2D) only the crosspeak for H at C-1 (Figure 2E), pointing
2
13
to the production of (1S)-(1- H,1- C)-1. Thus, the reprotonation
takes place from the Si-face in 6. The stereochemical course for
the reprotonation was also addressed during the investigation
of the recently characterized (+)-intermedeol synthase from
Streptomyces clavuligerus ATCC 27064 showing the same
14
15
1
4
9
1
4
9
6
2
5
H
10
10
5
2
3
8
2
3
8
3
4
9
8
1
0
1
6
15
12
5
12
7
7
19
7
6
11
13
outcome. Since the amino acid sequences of both terpene
11
15
H
H
OH
HO 14
14
1
3
11
synthases are not related (pairwise amino acid sequence
identity of 13.9%) the results show a case of convergent
evolution with regard to the product and even to the
stereochemical course, in which reprotonation of intermediate
6 has to take place from the opposite face as the attack of the
double bond at C-7. The regiochemistry of the deprotonation to
13
12
1
2
3
Figure 1: Main products of the terpene synthases characterized in this work.
Mechanistic characterization of STC4
The GC-MS analysis of the products obtained from an
incubation experiment with FPP and STC4 showed 1 as the
1
3
13
6
was surveyed by incubation of (12- C)FPP and (13- C)FPP
2
| J. Name., 2012, 00, 1-3
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Figure 3). The experiment with (12- C)FPP resulted in a
ARTICLE
1
3
(
View Article Online
DOI: 10.1039/C8OB02744G
1
3
product with a strongly enhanced C signal for the C-12 methyl
1
3
group in 1 (Figure 3A), while (13- C)FPP gave a product showing
a strong signal for the C-13 methylene group (Figure 3B),
pointing to a selective deprotonation at C-13. However, in both
spectra a minor signal corresponding to the other possible
position is visible (Figure 3), which suggests that also a small
fraction of the deprotonation is taking place at C-12. This
observation can be explained by an acting base that can reach
both methyl groups next to the cationic center in 5, but the C-
1
3 position is strongly favored.
Figure 3: 13C-NMR spectra resulting from the incubation experiments of STC4 with
different FPP isotopomers. A: (12-13C)FPP; B: (13-13C)FPP.
Mechanistic characterization of STC9
GC-MS analysis of the products obtained from STC9 and FPP showed
the highly selective production of -cadinene (2) (Figure S3). A
possible cyclization reaction starts from FPP, which is isomerized to
nerolidyl pyrophosphate (NPP) to allow a cisoid conformation
(Scheme 2). Abstraction of pyrophosphate and a 1,10-cyclization
Figure 2: Incubation experiment of STC4 with (6-13C)FPP. A: Cyclization reaction; B:
at C-1 in 1; C: 13C-NMR spectrum of (1- H,1-13C)-1, showing leads to formation of the (Z,E)-germacradienyl cation 10, that reacts
2
Representation of H

and H

an enhanced triplet as the signal for C-1, the signal near the solvent signal corresponds
to (6-13C)farnesol originating from non-enzymatic hydrolysis of (6-13C)FPP; D: Partial
by a 1,3-hydrid shift to the allylic cation 11. Direct 1,6-ring closure
leads to the cadinyl cation 12 and yields 2 after a final deprotonation.
 
HSQC spectrum of unlabeled 1, showing cross peaks of H and H at C-1; E: Partial HSQC
spectrum of labeled 1 from the incubation experiment, showing only the crosspeak for
14

H .
1
,10-
4
8
OPP-
cyclization
~
10
-OPP-
15
PPO
1
PPO 12
13
FPP
NPP
10
1
,3-H-~
H
H
1,6-
cyclization
-
H+
H
H
2
12
11
Scheme 2: Cyclization mechanism from FPP towards 2 catalyzed by STC9.
2
This mechanism was supported by incubation with (1,1- H
2
,11-
1
3
13
C)FPP to follow the 1,3-hydride shift. C-NMR analysis of the C
6 6
D
extract from the incubation showed a triplet for C-11, indicating a
2
13
direct H- C bond as expected for the proposed hydrogen migration
2
2
(
Figure 4A). Incubation with (1R)-(1- H)FPP and (1S)-(1- H)FPP
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followed by GC-MS analysis of the products showed in both cases an
View Article Online
DOI: 10.1039/C8OB02744G
increased molecular ion at m/z = 205. Moreover, the base peak of
2
the product from (1R)-(1- H)FPP was detected at m/z = 162, while
2
the product from (1S)-(1- H)FPP showed a base peak at m/z = 161
(Figure S4). Since the base peak corresponds to the fragment ion
after cleavage of the isopropyl group (Figure S4A), the deuterium
atom can be localized in the isopropyl group or within the core
structure, and can thus be used to follow deuterium migration.
Conclusively, the pro-S hydrogen at C-1 of FPP undergoes selectively
the shown hydride shift. The stereochemical fate of the geminal C-12
and C-13 methyl groups in FPP was investigated by incubations with
1
3
13
13
(
12- C) and (13- C)FPP. Analysis of the resulting C-NMR spectra
showed that each incubation led to the selective production of only
one isotopomer of 2 (Figures 4B and 4C), indicating that the 1,3-
hydride migration from 10 to 11 proceeds with strict face selectivity
with respect to the attack of the diastereotopic faces of the cationic
center in 10. An incubation experiment using FPP in D
produce a different isotopomer than in H O, as proven by GC-MS
analysis, pointing to a direct cyclization mechanism as depicted in
Scheme and disfavoring deprotonation-reprotonation
mechanism via germacrene D (Scheme S1).
An incubation experiment in D O has also been conducted for the
multi-product forming cadalane synthase MtTPS5 from Medicago
2
O did not
2
2
a
2
3
2
2
4
truncatula. For this enzyme the formation of the neutral
intermediate germacrene D and its reprotonation for further
cyclization seems likely, which is also reflected by the observation of
2
4
germacrene D as one of the products of the enzymatic reaction. The
(
(
–)--cadinene synthase from the bacterium Chitinophaga pinensis
Cp-CS) was also previously studied, including mechanistic aspects
2
1
with regards to the 1,3-hydride shift and the stereochemical fate of
2
0
the geminal methyl groups of FPP, showing the same results as for
STC9 in this work. Production of 2 was also shown for Omp5a and
Omp5b, two terpene synthases from the basidiomycete O. olearius,
9
when heterologously expressed in E. coli. However, no information
regarding the cyclization mechanism or the absolute configurations
of the products was obtained in this study. STC9 shows no close
sequence similarity to any of these enzymes. This represents again
an example of convergent evolution.
Figure 4: 13C-NMR analysis of incubation experiments of STC9 using different FPP
2
,11-13C)FPP; B: (12-13C)FPP; C: (13-13C)FPP. Peaks not marked
isotopomers. A: (1,1- H
2
with chemical shifts correspond to farnesol (hydrolysed FPP).
Mechanistic characterization of STC15
Recombinant STC15 produced mainly (+)-germacrene D-(4)-ol (3) in
in vitro experiments, but GC-MS analysis showed also considerable
amounts of -cadinene (2), -cadinene (13), -cadinene (14) and 6,
detected as its Cope rearrangement product -elemene (16) (Figure
S5). A cyclization mechanism that explains the formation of 3 and all
cadalane-products is very similar to the STC9 mechanism, including
FPP rearrangement to NPP, 1,10-ring closure, and a 1,3-hydride shift
to produce intermediate 11 (Scheme 3). This allylic cation can be
attacked by water, leading to production of 3. Alternative ring
closure to intermediate 12 and subsequent deprotonations yield
either -cadinene (13), -cadinene (14) or -cadinene (2). Notably,
4
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the formation of 6 does not require the isomerization to NPP and can for only one of the geminal methyl groups of 3 (FigureVisew5BArtaicnledO5nlCin)e,
proceed via direct 1,10-ring closure to the (E,E)-germacradienyl pointing to a strict stereochemical course DfoOrI:t1h0e.110,339-/hCy8dOrBid0e27s4h4iGft
cation (5) and deprotonation.
from 5.
H O
HO
2
3
15
6
-H+
1
,3-H-~
8
4
1
,10-
1
cyclization
13
4
1
-
OPP-
OPP 12
15
FPP
4
5
14
1
,10-
4
8
OPP-
cyclization
~
10
-OPP-
15
PPO
1
PPO 12
FPP
13
NPP
10
1
,3-H-~
H
H
H
b)
a)
b)
-
H+
H
H O
2
2
12
11
-
H+
-H+
a)
H
H
H
HO
13
14
3
Scheme 3: Cyclization of FPP towards 3, 2, 13, 14 and 6 catalyzed by STC15.
The formation of the main product 3 is also possible via 5, which can
undergo a 1,3-hydride shift to 15 followed by the attack of water.
2
13
This mechanism was supported by incubation of (1,1- H
with STC15 and subsequent NMR analysis, giving evidence for the
,3-hydride shift by a characteristic triplet for the signal of C-11 in 3
Figure 5A). A selective migration of the pro-S hydrogen in the
2
,11- C)FPP
Figure 5: 13C-NMR analysis of products obtained from incubation experiments of STC15
1
(
2
, 11-13C)FPP; B: (12-13C)FPP; C: (13-13C)FPP.
with different FPP isotopomers. A: (1,1- H
2
The unmarked signal in A corresponds to (11-13C)farnesol.
cyclization of 3 (Figure S6) and the byproducts 2 (same result as for 2
in STC9, Figure S4), 13 (Figure S7) and 14 (Figure S8) was shown by
This is the first report on a germacrene D-4-ol synthase, that
produces the (+)-enantiomer, while (–)-germacrene D-4-ol synthases
2
2
incubation of (1R)-(1- H)FPP and (1S)-(1- H)FPP and GC-MS analysis.
The stereochemical course of the hydride shift to produce 3 via both
shown starting conformations in Scheme 3 is the same (Scheme S2),
so no distinction regarding the productive starting conformation of
FPP for the cyclization to 3 can be made from these experiments. A
case of a mixed initial 1,10-cyclization of FPP and NPP is rare, but not
unprecedented, as it was also observed for the promiscuous -
2
1
from the bacteria Collimonas pratensis
and Streptomyces
2
6
citricolor were reported previously. Notably, the enzyme from C.
pratensis also produces -, - and -cadinene as side products that
are, just like the main product, the enantiomers of the identified
2
1
products of STC15.
2
5
humulene synthase from Abies grandis. An incubation experiment
in D O showed the same result as in H O and thus, as in the case of
Site-directed mutagenesis experiments
2
2
Although type I terpene synthases often show little overall sequence
similarity, there are some highly conserved motifs, like the aspartate
rich motif and the NSE/DTE-triad, that are crucial for magnesium
STC9, disfavors germacrene D as an uncharged intermediate that
would require reprotonation (Scheme S1). The incubations of STC15
1
3
13
with (12- C)FPP and (13- C)FPP showed a strongly enhanced signal
1
binding, and the RY pair and the pyrophosphate sensor forming
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hydrogen bond networks to the substrate’s diphosphate.2b Recently,
the alignment of a multitude of bacterial terpene synthase amino
View Article Online
DOI: 10.1039/C8OB02744G
3
c,27
acid sequences revealed new conserved residues,
most of which
putatively play a major structural role for the -domain fold. One
highly conserved residue is a tryptophan, appearing six amino acid
positions upstream of the RY pair. The corresponding residue was
previously targeted by site directed mutagenesis experiments with
2
8
pentalenene synthase and with cyclooctat-9-en-7-ol synthase
2
9
(CotB2).
For pentalenene synthase, exchange against
phenylalanine did not severely affect production, but gave
germacrene A (6) as a side product. For CotB2, the phenylalanine
variant showed a decreased production of cycloocat-9-en-7-ol, while
a glycine variant resulted in the formation of 3,7,18-dolabellatriene
as the main product.
All three terpene synthases surveyed in this work show a tryptophan
six positions upstream of RY (Figures S9-S11) and an exchange via site
directed mutagenesis of this residue to alanine and to phenylalanine
was carried out for each enzyme. In all three cases the alanine
variants gave no soluble protein from heterologous expressions in E.
coli. The effects of an exchange to phenylalanine were different in
each tested enzyme. For STC4 W335F, the production of 1 was
completely abolished and 6 was observed as main product, as shown
by GC-MS detection of its Cope rearrangement product 16 (Figure 6).
This finding suggests a strong effect of W335 on the stabilization of
cation 7 by cation--interactions that may be important for
downstream reactions from 6 (Scheme 1). Such a stabilization is less
Figure 7: Relative amount of produced 2 of STC 9 (set to 100%) and its tested variants as
determined by comparative incubations followed by GC-MS analysis. All experiments
were carried out in triplicate.
Since Cp-CS produced the same enantiomer of (–)-2 via the same
cyclization mechanism as STC9, homology models of both enzymes
based on the crystal structure of selinadiene synthase from
2b
Streptomyces pristinaespiralis (PDB code 4OKM, SdS) were
constructed and compared to a homology model of the (–)--
3
0
pronounced for phenylalanine. However, 7 and 8 are still observed
in the W335F variant. A possible explanation is that these
compounds are formed non-enzymatically from 6, a reaction that
32
cadinene synthase from S. clavuligerus (Sc-CS), based on the same
crystal structure (Figures S12 – S15). (–)--Cadinene (13) produced
by Sc-CS exhibits an absolute configuration corresponding to the
absolute configuration of 2 and its cyclization was also shown to
3
1
smoothly proceeds by mild acid catalysis in solution or on silica gel.
The STC9 W314F variant showed the same product specificity and
the same activity (Figure 7) as the wild type enzyme, so W314 can be
substituted by Phe for efficient catalysis by this enzyme. In the case
of STC15, the heterologous expression of the W314F variant failed to
produce soluble protein.
32
proceed via a 1,3-hydride shift of the pro-S proton. Only the last
deprotonation step to produce 13 is different (Scheme 3) and thus
polar residues from the three synthases that point into the active site
and could interfer with the mode of deprotonation were compared.
The most promising residues were C311 and Y315 in STC9, which are
positioned next to the conserved W314 (Figures S10 and S12). The
corresponding positions are occupied by Asn and Ser in Cp-CS
(
Figure S13) and by His and Gly in Sc-CS (Figure S14). While there
are two polar residues, that can provide hydrogen bonds in the two
-cadinene synthases, a basic residue and a glycine occur in Sc-CS,

the first of which could account for the alternative deprotonation of
cationic intermediate 12 towards 13 instead of 2. Variants of STC9
with exchange of a single residue were constructed (C311H, C311N
and C311S, and Y315G and Y315S). The product spectrum of all
variants was unchanged, still showing selective production of 2, but
the production was strongly decreased in the C311H variant (3%) and
moderately affected in the C311N variant (45%) (Figure 7). For the
C311S, Y315G and Y315S variants the production was comparable to
WT STC9. When the Y315 and C311 mutations were combined in one
enzyme variant, compound 2 was still the only detectable product,
but the relative enzyme activity dropped significantly, ranging from
1
% production by the C311N/Y315G variant to 30% by the
C311N/Y315S variant. These mutagenesis experiments show the
dependency of efficient catalysis by STC9 on C311, only exchange
with its oxygen analog Ser retained almost the same productivity
Figure 6: GC-MS analysis of incubations of STC4 incubations using FPP. A: wild type; B:
W335F variant. Compound 17 is a non-enzymatic degradation product of FPP and also
occurs without enzyme.
(80%) than in the WT enzyme. Y315 exchange does not seem to be
6
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ARTICLE
critical, but when it was exchanged by Gly or Ser in combination with Conflicts of interest
View Article Online
DOI: 10.1039/C8OB02744G
C311 mutations the productivity dropped more severely in all tested
There are no conflicts to declare.
cases. Since both original residues C311 and Y315 are rather
3
3
hydrophobic, replacement by more hydrophilic residues may
disturb the hydrophobic cavity and thus hamper catalysis, but
detailed insights into the particular roles of these residues will
require structural data of STC9.
Acknowledgements
This work was supported by the DFG with grant DI1536/9-1 and
the CRC 1127 ChemBioSys. We thank M. T. Poulsen (University
of Copenhagen, Denmark) and Z. W. de Beer (FABI, University
of Pretoria) for supporting our field work.
Conclusions
We characterized three terpene synthases from the termite
associated fungus Termitomyces, an interesting species with largely
untapped secondary metabolism, as (+)-intermedeol synthase
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