An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma

Article abstract of DOI:10.1002/anie.201907964

The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring-opening reaction at a terpene-cyclase-derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp. is described that can convert farnesyl diphosphate (FPP) directly via a remarkable skeletal rearrangement into trichobrasilenol, a new brasilane sesquiterpene with one additional Me group equivalent compared to FPP. A mechanistic hypothesis for the formation of the brasilane skeleton is supported by extensive isotopic labelling studies.

Full text of DOI:10.1002/anie.201907964

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Accepted Article
Title: An Unusual Skeletal Rearrangement in the Biosynthesis of the
Sesquiterpene Trichobrasilenol from Trichoderma
Authors: Keiichi Murai, Lukas Lauterbach, Kazuya Teramoto, Zhiyang
Quan, Lena Barra, Tsuyoshi Yamamoto, Kenichi Nonaka,
Kazuro Shiomi, Makoto Nishiyama, Tomohisa Kuzuyama, and
Jeroen Sidney Dickschat
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201907964
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10.1002/anie.201907964
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An Unusual Skeletal Rearrangement in the Biosynthesis of the
Sesquiterpene Trichobrasilenol from Trichoderma
Keiichi Murai,[a] Lukas Lauterbach,[b] Kazuya Teramoto,[c] Zhiyang Quan,[b] Lena Barra,[b] Tsuyoshi
Yamamoto,[d] Kenichi Nonaka,[d] Kazuro Shiomi,[d] Makoto Nishiyama,[c,e] Tomohisa Kuzuyama*[a,e] and
Jeroen S. Dickschat*[b]
Abstract: The skeletons of some classes of terpenoids are unusual,
because they contain a larger number of Me groups (or their
biosynthetic equivalents such as olefinic methylene groups,
hydroxymethyl groups, aldehydes or carboxylic acids and their
derivatives) than provided by their oligoprenyl diphosphate precursor.
This is sometimes the result of an oxidative ring opening reaction at a
terpene cyclase derived molecule containing the regular number of
Me group equivalents, as observed for picrotoxan sesquiterpenes. In
this study a sesquiterpene cyclase from Trichoderma is described that
can convert farnesyl diphosphate (FPP) directly via a remarkable
cyclases (TCs) that ionise their substrate by diphosphate
abstraction or by protonation.[1-5] The initially formed cation can
make use of its inherent reactivity leading usually to a polycyclic
skeleton with several stereogenic centres.[6] In these reactions the
main tasks of the enzyme are to control the substrate
conformation, to trigger the start of the reaction by the ionisation
step, to provide a hydrophobic cavity essentially free of water to
avoid premature cation quenchings, and to terminate the reaction
by a strictly controlled deprotonation or addition of water to the
latest cationic intermediate. For many of the known polycyclic
skeletons a possible mechanism for the formation from their
corresponding precursor seems obvious, but sometimes only
isotopic labelling experiments can unravel surprising
rearrangement steps or are required to distinguish between
different alternatives.[7-9] The diversity of skeletons is further
enlarged by irregular couplings of DMAPP and IPP that can help
to explain unusual terpene skeletons,[10] but in other cases
skeletal rearrangements of an acyclic (regular) precursor must be
assumed. Particularly interesting are systems, in which the
number of Me groups or their equivalents such as olefinic
methylene groups exceeds the number of Me groups in the
precursor. For example, the picrotoxan sesquiterpene alkaloid
dendrobine (1) from Dendrobium nobile,[11,12] a compound with
antipyretic, hypertensive and convulsant activity, contains five
such Me group equivalents (Figure 1). Its formation from FPP,
containing four Me groups, has been demonstrated by the
incorporation of labelling from tritiated farnesol fed to D. nobile.[13]
The conversion of regiospecifically labelled copaborneol (2) into
tutin (3) by Coriaria japonica demonstrated that the additional Me
group equivalent in picrotoxans is a result of a ring opening
reaction of 2 in a post-TC step,[14] while 2 itself can be rationalised
as a regular sesquiterpene cyclase (STC) product (Scheme S1).
skeletal rearrangement into trichobrasilenol,
a new brasilane
sesquiterpene with one additional Me group equivalent compared to
FPP. A mechanistic hypothesis for the formation of the brasilane
skeleton is supported by extensive isotopic labelling studies.
With an estimated number of more than 80,000 different
compounds, terpenoids represent the largest class of natural
products.[1] Their basic carbon skeletons derive from just a few
acyclic precursors made by regular coupling of the monomers
dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate
(IPP), including geranyl (GPP), farnesyl (FPP), geranylgeranyl
(GGPP) and geranylfarnesyl diphosphate (GFPP). These
precursor molecules are converted by the action of terpene
[a]
[b]
Keiichi Murai,[+] Prof. Dr. Tomohisa Kuzuyama
Graduate School of Agricultural and Life Sciences
The University of Tokyo
1-1-1 Yayoi, Bunkyu-ku, Tokyo 113-8657, Japan
E-mail: utkuz@mail.ecc.u-tokyo.ac.jp
Lukas Lauterbach,[+] Dr. Zhiyang Quan, Dr. Lena Barra, Prof. Dr.
Jeroen S. Dickschat
Kekulé-Institute for Organic Chemistry and Biochemistry
University of Bonn
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany
E-mail: dickschat@uni-bonn.de
[c]
[d]
Dr. Kazuya Teramoto, Prof. Dr. Makoto Nishiyama
Biotechnology Research Center
The University of Tokyo
1-1-1 Yayoi, Bunkyu-ku, Tokyo 113-8657, Japan
Dr. Tsuyoshi Yamamoto, Dr. Kenichi Nonaka, Prof. Dr. Kazuro
Shiomi
Kitasato Institute for Life Sciences
Kitasato University
5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Prof. Dr. Makoto Nishiyama, Prof. Dr. Tomohisa Kuzuyama
Collaborative Research Institute for Innovative Microbiology
The University of Tokyo
1-1-1 Yayoi, Bunkyu-ku, Tokyo 113-8657, Japan
These authors contributed equally to this work.
[e]
[+]
Figure 1. Examples of sesquiterpenoids with five Me group equivalents.
Supporting information for this article is given via a link at the end of
the document.
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The marasmane derivative isovelleral (4) also contains five Me
group equivalents and is a strongly antifungal and antibacterial
sesquiterpenoid with a pungent taste from the fungus Lactarius
vellereus,[15] but the biogenesis of 4 or related compounds of the
marasmane type has not been investigated so far. Also brasilane
sesquiterpenes such as the antibacterial compound xylarenic acid
(5) exhibit an extra Me group equivalent compared to FPP.[16] We
have addressed the intricate, and despite all the accumulated
work on terpene biosynthesis still unsolved mechanistic problem
of the formation of such additional Me groups by the identification
of a brasilane STC from Trichoderma that directly extrudes a fifth
Me group equivalent from the FPP chain during the terpene
cyclisation cascade.
The genome of Trichoderma atroviride FKI-3849[17] was
sequenced and found to encode at least seven type I TCs (named
TaTC1–7). The gene of one of these enzymes (TaTC6, accession
number LC484924) that is not clustered with other biosynthetic
genes and whose gene product is phylogenetically distant from
other characterised fungal TCs (Figure S1) was cloned and
heterologously expressed in Aspergillus oryzae, resulting in the
formation of a new product (Figure S2). The purified protein
obtained by heterologous expression in Escherichia coli
BL21(DE3) (Figure S3) exhibited all highly conserved motifs that
are required for substrate binding and functionality (Figures S4
and S5) and converted FPP into a mixture of sesquiterpene
hydrocarbons and alcohols (Figure S6), while GPP, GGPP and
GFPP were not accepted. The main product was purified by silica
column chromatography and identified by extensive NMR
spectroscopy (Table S3 and Figures S7 – S13) as a new
sesquiterpene alcohol for which we suggest the name
trichobrasilenol (6, Scheme 1A). The phylogenetic tree (Figure
S1) shows that closely related enzymes are present in several
other Trichoderma strains (cf. also Figure S14), and heterologous
expression of the corresponding gene from Trichoderma reesei
QM6a in Aspergillus oryzae yielded the same compound 6 (Figure
S15), suggesting the same function for all these related enzymes.
Figure 2. The absolute configuration of 6. Partial HSQC spectra of A) unlabelled
6, B) labelled 6 obtained from DMAPP and (E)-(4-13C,4-2H)IPP, and C) labelled
6 obtained from DMAPP and (Z)-(4-13C,4-2H)IPP (Scheme 1B). Important NOE
correlations for interpretation of the results are specified, additional NOE
correlations are shown in Scheme 1.
Scheme 1. Trichobrasilenol (6). A) Structure elucidation (bold: COSY, single
headed arrows: key HMBC, double headed arrows: key NOESY correlations).
B, C) Isotopic labelling experiments to solve the absolute configuration of 6. D,
E, F) Isotopic labelling experiments to follow hydride shifts in the biosynthesis
of 6.
The absolute configuration of
6
was resolved using
enantioselectively deuterated FPP isotopomers. Their conversion
with TaTC6 resulted in stereoselectively deuterated carbons of 6
with known configuration. The absolute configuration was then
correlated by solving the relative orientation of the other
stereocentres to the incorporated deuterium. An additional 13C-
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label at the deuterated carbon allowed for sensitive HSQC
analysis of the products obtained from small scale reactions
without the need of compound purification. Specifically, (E)- and
(Z)-(4-13C,4-2H)IPP[18] were used to elongate DMAPP using the
FPP synthase (FPPS) from Streptomyces coelicolor A3(2),[19]
followed by cyclisation with TaTC6, resulting in the incorporation
of labellings at C4 and C8 (Scheme 1B). The HSQC spectra of
the labelled products (Figure 2) together with the assignments of
the diastereotopic hydrogens at C4 based on the NOESY
spectrum (Table S3 and Figure S13) pointed to the absolute
configuration of 6 as shown in Scheme 1. Similarly, DMAPP was
elongated with (R)- and (S)-(1-13C,1-2H)IPP[20] catalysed by FPPS
for cyclisation by TaTC6 (Scheme 1C). The HSQC spectra of the
products labelled at C1 and C5 indicated the same absolute
configuration for 6 (Figure S16). The interpretation of these
experiments is based on the known stereochemical course for the
addition of IPP to DMAPP and GPP in the elongation to FPP,[21]
and on the assumption of an inversion of configuration at C1 of
FPP in the initial cyclisation, as observed for other TCs.[22]
TaTC6 towards 6 (Scheme 2B) starts from FPP by abstraction of
diphosphate and 1,11-cyclisation. To avoid secondary cations as
intermediates a concerted reaction with simultaneous 1,2-hydride
migration and double ring closure to A can be assumed, which is
followed by another 1,2-hydride shift to B. A third 1,2-hydride
transfer leads to C that can be attacked by water with a
remarkable rearrangement through the transition state D‡,
involving another simultaneous hydride shift, cyclopropane ring
opening, ring contraction and double bond installation, ultimately
leading to 6. The side product 7 can be explained by 1,11-
cyclisation of FPP with direct deprotonation from C9, while 8 and
9 require additional 2,10-cyclisation. The compounds 10 – 13 may
arise through very similar reactions as 6, but their stereochemistry
at Me-15 is different, requiring a different stereochemical course
of the cyclisation cascade.
Figure 3. NMR spectra of the products obtained in labelling experiments to
follow hydride shifts in the biosynthesis of 6. Partial 13C-NMR spectra of
unlabelled 6 (top) and of labelled 6 (bottom) obtained from A) (3-13C,2-2H)FPP,
B) (2-2H)GPP and (2-13C)IPP, and C) (2-13C,1,1-2H2)DMAPP and (2-13C)IPP.
Scheme 2. A) Identified side products of TaTC6. B) Cyclisation mechanism from
FPP to 6. Compound numbering for 6 shows the origin of each carbon from FPP.
Also several side products were identified by GC/MS (Scheme 2A,
Table S5), including -humulene (7), caryophyllene (8), 2-epi-
caryophyllene (9), african-3-ene (10) and african-1-ene (11),
while isoafricanol (12) and pristinol (13) were identified by
comparison to authentic standards obtained with recently
characterised STCs from Streptomyces malaysiensis and
Streptomyces pristinaespiralis.[23,24] All these compounds are
formed from FPP by an initial 1,11-cyclisation, suggesting that this
may also be the case for 6. A proposed cyclisation mechanism for
This mechanistic hypothesis, explaining the direct formation of a
fifth Me group equivalent in a TC catalysed reaction, was
investigated in a series of isotopic labelling experiments. The
incubation of all fifteen isotopomers of (13C)FPP[25] with TaTC6
resulted in the incorporation of labelling into the expected
positions in all cases (Figure S17), which especially supported the
proposed rearrangement of the C6-7-8-9-10 portion of FPP in the
step from C via D‡ to 6.
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The hydride migration from A to B was followed with (3-13C,2-
2H)FPP,[24] resulting in a slightly upfield shifted strong triplet for C3
of 6 in the 13C-NMR spectrum (Scheme 1D, Figure 3A). The third
hydride migration from B to C was investigated through the same
strategy using (2-2H)GPP[26] and (2-13C)IPP[27] that were coupled
with FPPS to (2-13C,6-2H)FPP. Enzymatic conversion with TaTC6
yielded a product with a direct 13C-2H bond as indicated by a
strongly enhanced triplet for C2 in the 13C-NMR spectrum
(Scheme 1E, Figure 3B). The remaining two hydride shifts from
FPP to A and from C via D‡ to 6 were addressed simultaneously
in an experiment with (2-13C,1,1-2H2)DMAPP and (2-13C)IPP that
were coupled with FPPS to yield (2,6,10-13C3,9,9-2H2)FPP. Its
conversion by TaTC6 into labelled 6 yielded three enhanced
signals for the labelled carbons (Scheme 1F, Figure 3C). The
signal for C2 showed a very small upfield shift explainable by a
deuterium at a neighbouring carbon and a doublet coupling with
C6. The signal for C6 exhibited a typical upfield shift for a directly
bound deuterium, also evident from the triplet coupling in addition
to the doublet coupling with C2. Finally, C10 was also connected
to deuterium as evident from the upfield shift and the triplet
coupling. Taken together, these data supported both hydride
shifts from FPP to A and from C to 6.
This work was supported by the DFG (DI1536/7-1) and JSPS
KAKENHI (16H06453).
Keywords: biosynthesis • enzyme mechanisms • isotopes •
NMR spectroscopy • terpenes
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In summary, we have identified two representatives from a clade
of STCs from Trichoderma that produce the new sesquiterpene
alcohol trichobrasilenol with an additional Me group equivalent in
comparison to FPP. The mechanism of its formation was
addressed using isotopically labelled precursors, demonstrating
the extrusion of this additional Me group equivalent in form of a
hydroxymethyl group from an internal methylene group of FPP in
a notable rearrangement step along the cationic cyclisation
cascade to 6. This main finding of our study represents an
unprecedented reaction in terpene biosynthesis most likely in a
concerted process, as there is no good explanation via a stepwise
procedure (cf. Scheme S2 with a detailed discussion in the SI). A
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dependent methylation, but in this case three more Me groups
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group equivalents into terpenes, and with this study we provide
the first mechanistic investigation of a TC that catalyses carbon
extrusion by a complex skeletal rearrangement.
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COMMUNICATION
Trichobrasilenol was identified as an
unusual sesquiterpene alcohol made
by a sesquiterpene cyclase from
Trichoderma. The mechanism of the
product formation was studied using
isotopically labelled substrates, with a
special emphasis on a complex
rearrangement step that leads to the
extrusion of a hydroxymethyl group
from an internal methylene group of
FPP.
Keiichi Murai, Lukas Lauterbach,
Kazuya Teramoto, Zhiyang Quan, Lena
Barra, Tsuyoshi Yamamoto, Kenichi
Nonaka, Kazuro Shiomi, Makoto
Nishiyama, Tomohisa Kuzuyama* and
Jeroen S. Dickschat*
Page No. – Page No.
An Unusual Skeletal Rearrangement
in the Biosynthesis of the
Sesquiterpene Trichobrasilenol from
Trichoderma
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