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Cite this: Nat. Prod. Rep., 2012, 29, 60
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Sesquiterpene synthases: Passive catalysts or active players?
David J. Miller and Rudolf K. Allemann*
Received 29th July 2011
DOI: 10.1039/c1np00060h
Covering: up to July 2011
Sesquiterpene synthases catalyse the metal dependent turnover of farnesyl diphosphate to generate
a class of natural products characterised by an enormous diversity in structure, stereochemistry,
biological function and application. It has been proposed that these enzymes take a passive role in the
reactions they catalyse and that they serve mostly as stereochemical templates, within which the
reactions take place. Here, recent research into the structure and function of sesquiterpene synthases
and the mechanisms of the reactions that they catalyse will be reviewed to suggest that these fascinating
enzymes play multifaceted active roles in what are arguably the most complex biosynthetic reactions.
1 Introduction
2 Metal ion binding and diphosphate cleavage
3 Carbocationic reaction cascades
3.1 Site directed mutagenesis studies
3.2 Aza-analogues
3.3 Substrate analogues
4 Acid/base catalysis
5 Conclusions
6 Acknowledgments
7 References
1 Introduction
With tens of thousands of characterised members throughout all
forms of life, terpenoids are the largest and most structurally
diverse class of natural products.1,2 The chemically most
intriguing contribution to the generation of this diversity is made
by terpene synthases, enzymes that convert their linear isoprenyl
diphosphate substrates into terpenoid hydrocarbons in what are
perhaps the most complex biosynthetic reactions. They catalyse
the production of a multitude of compounds often containing
several stereocentres via carbocationic reaction cascades that
involve changes to the connectivity and hybridisation of up to
half the carbon atoms of the substrate.
Excellent reviews have been published on the biosynthesis of
monoterpenes,3–6 diterpenes5,7 and triterpenes;8–10 the focus here
will be on sesquiterpenes and particularly on sesquiterpene syn-
thases. Sesquiterpene synthases catalyse, within a shared three-
dimensional fold, the conversion of farnesyl diphosphate (FDP,
1a) into more than 300 different sesquiterpenes, all with the
School of Chemistry, Cardiff University, Main Building, Park Place,Cardiff, CF10 3AT, United Kingdom. E-mail: allemannrk@cardiff.ac.uk;Fax: (+41) 29-2087-4030
60 | Nat. Prod. Rep., 2012, 29, 60–71
shared formula C15H24. In the past, monographs have been
dedicated to these enzymes from experimental and theoretical
perspectives.3,11–13 We will be examining the role played by
sesquiterpene synthases in this masterpiece of combinatorial
chemistry from a mechanistic physical organic chemistry view-
point and will aim to describe the general principles shared by all
sesquiterpene synthases for catalysis.
The catalytic mechanisms of sesquiterpene synthases can be
divided into two phases; the metal ion dependent binding of the
substrate and cleavage of the C–O bond to generate an ion pair
between diphosphate and a farnesyl cation is followed by
a precise carbocationic reaction cascade to the final reaction
products. It has previously been suggested that after the initial
ionisation, the enzymes act as passive catalysts that simply
provide templates to chaperone the intermediates along the
reaction pathway. We will suggest a more active role for these
enzymes in catalysis, showing that they are intimately involved in
the chemistry at all stages of the reaction pathway.
The review will be divided into three sections: (i) investigation
of metal ion, substrate binding and generation of the initial
carbocation, (ii) analysis of the role of the enzyme in FDP
folding, cation stabilisation and coordination of the intermedi-
ates and transition states of the cyclisation cascade, and (iii)
study of the nature of putative acids and bases in the enzymes’
active sites.
2 Metal ion binding and diphosphate cleavage
The formation of sesquiterpenes is initiated by the divalent metal
(usually Mg2+) dependent cleavage of diphosphate (PPi) from
FDP; three metal ions are bound at the top of the active site and
coordinate diphosphate prior to cleavage. Metal coordination is
most easily visualised through structural analysis. At the time of
writing the protein databank (http://www.pdb.org) contains the
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crystal structures of seven distinct sesquiterpene synthases that
have been solved in forty two forms; both wild type and mutant
enzyme structures have been reported in unliganded forms and in
complex with various combinations of metal ions, diphosphate,
FDP, substrate analogues and/or organic cation mimics. All
sesquiterpene synthases contain the class I terpene synthase
a-helical fold, within which the reactions takes place; plant
sesquiterpene synthases also contain a catalytically silent class II
terpene synthase fold (Fig. 1).4 The most conserved motifs in the
class I fold are the ‘DDXXD/E’-motif, which is located on helix
D and binds Mg2+A and Mg2+C , and the ‘DTE/NSE’ motif on helix
H, which binds Mg2+B .4 The recently solved crystal structure of
(+)-d-cadinene (22) synthase from Gossypium arboreum (GA-
DCS) (Fig. 1) revealed that the DTE motif in this enzyme is
absent and instead takes the form of a second DDXXD motif
more reminiscent of the presumed ancestral isoprenyl
Fig. 1 Sketches of the three most recently solved X-ray single crystal stru
Streptomyces coelicolor A3 (EIZS) in complex with Mg2+3 , PPi and benzyltrim
synthase from Gossypium arboreum (GA-DCS) in complex with 3 Mg2+ and 2F
in yellow); C: aristolochene (11) synthase from Aspergillus terreus (AT-AS) in
David J: Miller
David Miller studied chemistry
at the University of Oxford
(1993). He obtained his PhD
(1997) in bioorganic chemistry
under the tutelage of Prof. Tim
Bugg at the University of
Southampton developing inhibi-
tors for enzymes involved in
peptidoglycan biosynthesis.
Post-doctoral positions followed
at the University of St. Andrews
and the University of Birming-
ham under Prof. David Gani and
Prof. Rudolf Allemann. He was
appointed as a research fellow in
the School of Chemistry at
Cardiff in 2007. His research interests include terpene biosynthesis,
synthetic biology and medicinal chemistry.
This journal is ª The Royal Society of Chemistry 2012
diphosphate synthases.14 (+)-Germacrene D (26) synthase (GDS)
from Solidago canadensis also has an altered metal binding motif,
although in this case, it is the first DDXXD motif that takes the
form of NDTYD.15 Nevertheless, the two metal ion binding sites
in all known sesquiterpene synthases are responsible for coor-
dination of three putative metal cations and it is these that
coordinate the diphosphate group of the substrate. The binding
of the three magnesium ions is highly ordered and triggers the
closure of the active site, in which the farnesyl chain adopts
a reactive conformation sheltered from water.4,16–26
The crystal structures of (+)-aristolochene (11) synthases from
Penicillium roquefortii (PR-AS)27 and Aspergillus terreus19 (AT-
AS) (Fig. 1) offer an opportunity to compare two separately
evolved sesquiterpene synthases that generate the same product.
A shared active site contour for the two enzymes has been
revealed.19 A series of AT-AS structures were determined in the
ctures of sesquiterpene synthases. A: epi-isozizaene (17) synthase from
ethyl ammonium cation (BTAC, 51) (pdb 3KB9);23 B: (+)-d-cadinene (22)
-FDP (pdb 3G4F)14 (the catalytically silent class II-like domain is shown
complex with FDP (pdb 3BNX).21
Rudolf K: Allemann
Rudolf K Allemann studied
physics and chemistry at the
Eidgen€ossische Technische
Hochschule in Zurich (ETH-
Z), where he also received his
PhD after studies at Harvard
University and ETH-Z. He then
moved to the MRC-National
Institute for Medical Research
as a Postdoctoral Research
Fellow of the Royal Society
(London) and the Swiss
National Science Foundation.
After his habilitation at ETH-Z,
he took up positions at the
University of Birmingham,
ETH-Lausanne and Cardiff University, where he holds a Distin-
guished Research Professorship. The research of the Allemann
laboratory focuses on enzyme mechanism, catalysis and design.
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presence of the substrate and various fluorinated FDP
analogues.21 These crystals, which comprised the enzyme in
a tetrameric structure bound to Mg2+, PPi and FDP analogues in
various combinations provided a series of snapshots of the
enzyme in both open and closed form depending upon quater-
nary structure/crystal packing effects and the coordination state.
These structures led to the prediction of a sequence of binding
events for catalysis by AT-AS. Binding of the substrate to the
open form of the enzyme in the absence of metal ions is followed
by the coordination of Mg2+B and Mg2+C and active site closure;
the reactive conformation of FDP is formed after Mg2+A binds to
initiate the reaction cascade. Aristolochene (11), Mg2+A and Mg2+Crelease are accompanied by active site opening followed by
release of the third magnesium, Mg2+B , and diphosphate to
complete the reaction cycle.
Recently, a crystallographic analysis of epi-isozizaene (17)
synthase (EIZS) from Streptomyces coelicolor A3(2) (Fig. 1)
showed similar conformational changes upon binding of
ligands.23 The structure was solved in complex with three Mg2+
ions, PPi and benzyltrimethyl ammonium cation (BTAC, 51,
Fig. 2) as well as in complex with Hg2+; in addition, a structure of
the ligand-free EIZS with a mutation to the DDXXD motif was
also obtained. This work revealed a significant conformational
change of helix G to cap the active site upon metal binding. The
structure of trichodiene (20) synthase from Fusarium sporo-
trichioides (FS-TS) in complex with Mg2+, PPi and BTAC
showed similar conformational changes upon binding to metal
ions.22 In all these cases, it is the combination of binding to three
Mg2+ and PPi (FDP in the actual reaction) that triggers closure of
the active site, followed by the cleavage of the C–O bond in FDP.
Clearly, such a coordinated sequence of molecular events to
achieve the reactive conformation of the substrate must be
considered active enzyme participation.
Cation formation and carbocation cyclisation are of course
not separate but intimately linked events. For example, mutation
of the D100DSKD motif of FS-TS to E100DSKD resulted in an
increase in the active site volume and hence a more promiscuous
product spectrum indicating that metal ion binding and overall
active site contours are intimately linked.17 A site directed
mutagenesis (SDM) study aimed at investigating the role of the
NSE motif (typical of bacterial/fungal derived enzymes) in FS-
TS, where it was sequentially converted to a plant-like DTE
motif, showed a gradual reduction of product fidelity and cata-
lytic activity of the enzyme.20 Finally, Arg 304 of FS-TS forms
a hydrogen bond to PPi to stabilise the anion in the active site
and to ensure formation of the correct active site contour.18
These observations underline the notion that metal binding is
central to the fidelity of FS-TS catalysis.25,26,28
Recent work has shown that cation generation by sesquiter-
pene synthases need not necessarily precede the cyclisation
reactions, but the two processes often occur in a concerted
fashion. Interrogation of the mechanism of action of PR-AS
using 12,13-difluoro-farnesyl diphosphate (12,13-FF-FDP, 1n,
Fig. 4) showed that this compound was a competitive inhibitor of
PR-AS (vide infra for an explanation of the effects of fluoro
substituents).29 The fluorine atoms of 1n are distant from C1 and
should not inhibit formation of the farnesyl cation. Linear
difluoro-farnesene analogues were formed upon incubation of 1n
with the mutant enzyme F112A-PR-AS suggesting that in the
62 | Nat. Prod. Rep., 2012, 29, 60–71
absence of the phenyl ring of Phe 112 a farnesyl cation was
formed followed by proton loss to generate the farnesene prod-
ucts. This indicated that the role of Phe 112 in PR-AS is (together
with other bulky residues in the active site) to direct the distal
double bond of FDP into a reactive conformation to promote the
formation of germacrenyl cation (4) directly from FDP through
an SN2 like reaction and not via a farnesyl cation.29 This is
consistent with the observed inversion of configuration at C1
when (R)- and (S)-1-[2H]-FDP (1b and 1c, Fig. 3) were tested as
substrates of this enzyme.30 In further corroboration of the direct
displacement of PPi, 2-fluoro-farnesyl diphosphate (2F-FDP, 1k)
was turned over efficiently by PR-AS to a fluoro-germacrene A
analogue.31 Interestingly, 2F-FDP has been reported not to be
a substrate for tobacco 5-epi-aristolochene (12) synthase
(TEAS), an enzyme that also proceeds through the germacrenyl
cation (4), suggesting that a farnesyl cation is an intermediate in
the production of 12 and that the presence of fluorine prevents its
formation in this case. PR-AS plays a crucial active role in
determining the nature of the cation that is formed upon cleavage
of PPi – the catalysed reaction does not always proceed through
farnesyl cation and beyond for all sesquiterpene synthases.
Isomerisation of FDP to nerolidyl diphosphate (NDP 3) to
facilitate the production of products arising from a-bisabolyl (6),
cycloheptenyl (7), helminthogermacrenyl (8) or cis-humulenyl (9)
cations (Scheme 1) has been a difficult process to observe
experimentally. A study of the pre-steady state kinetics of FS-TS
revealed that formation of NDP is slow but once formed it reacts
onwards too quickly for NDP to be observed experimentally.32 A
recent study has revealed a single residue switch in the maize
terpene synthase 10 (TPS10) for NDP formation. TPS10
produces (mostly) a mixture of E-b-farnesene (30) and E-a-ber-
gamotene (21).33 The farnesene product arises from elimination
of H+ and PPi from FDP but the second product arises from
a downstream reaction after isomerisation to NDP. SDM of
TPS10 showed that Leu 356 controls the rate of isomerisation
from trans to cis farnesyl cations.33
3 Carbocationic reaction cascades
Following cleavage of PPi and the generation of the initial car-
bocation, sesquiterpene synthases steer the reaction through
often complex cyclisation cascades that contain additional car-
bocationic as well as neutral intermediates. In contrast to
promiscuous sesquiterpene synthases, such as g-humulene (27)
(GHS) and d-selinene (14) synthase (DSS) from Abies grandis34
or the recently discovered cadalane producing enzyme MtTPS5
from Medicago truncatula,35 high fidelity enzymes like AT- and
PR-AS or GA-DCS differentiate rigorously against alternative
reaction paths that will normally be of similar energies.
Directing the reaction along a specific reaction coordinate is
achieved through the provision of an active site template largely
made up of aromatic and aliphatic residues, where the enzyme’s
active site surface contour aligns orbitals in productive spatial
orientations, and through the stabilisation of the mostly carbo-
cationic intermediates and transition states. The latter effect has
been studied using a variety of experimental approaches,
including SDM of aromatic amino acid residues, the use of aza-
analogues, ammonium containing species, which mimic carbo-
cationic intermediates and substrate analogues designed to
This journal is ª The Royal Society of Chemistry 2012
Scheme 1 Early steps in the formation of cyclic sesquiterpenoids from FDP (1a) catalysed by sesquiterpene synthases and some of the resulting
products discussed in the text. Loss of H+ and PPi from FDP (1a) or NDP (3) can also lead to the linear farnesenes.
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intercept the reaction at specific points or to examine the regio-
and/or stereochemistry of specific steps in the catalytic cycle.
3.1 Site directed mutagenesis studies
PR-AS contains four aromatic residues in its active site (Tyr 92,
Phe 112, Phe 178 and Trp 334) and the influence of these have
been analysed in a series of SDM experiments.36–40 Mutants of
Trp 334 produce varying amounts of (�)-germacrene A (10), the
putative intermediate of PR-AS catalytic activity.41 This result
was interpreted as evidence that Trp 334 stabilises the positive
charge on the eudesmane cation, the species that is formed upon
protonation of germacrene A (10).38 Replacement of Trp 334
with para-substituted phenylalanines of increasing electron-
withdrawing properties led to a progressive accumulation of 10
This journal is ª The Royal Society of Chemistry 2012
with a good correlation with the interaction energies of simple
cations, such as Na+, with substituted benzenes.42 These results
provide compelling evidence for the importance of Trp 334 in
cation stabilization during the energetically demanding trans-
formation of the neutral germacrene A to eudesmane cation in
PR-AS catalysis. Tyr 92 may, in part, play a similar role to that
played by Trp 334 since incubation of FDP with PR-AS-Y92V
gave a similar product profile to that observed for PR-AS-
W334V.39 In addition, the bulkiness of Tyr 92 clearly contributes
to the accurate folding of the substrate in the active site; a series
of replacements of Tyr 92 revealed that the amounts of aristo-
lochene (11) increased with the van der Waals volume of the side
chain. When the bulkiness of residue 92 was reduced, linear
farnesenes were observed as the main products.43 While these
results suggest that cyclisation of FDP might proceed through
Nat. Prod. Rep., 2012, 29, 60–71 | 63
Scheme 2 Bifurcation in sesquiterpenoid cyclisation pathways. Through
the alterations of active site residues, tobacco 5-epi-aristolochene syn-
thase (TEAS) andHysocyamus premnaspirodiene synthase (HPS) can be
tuned to transform the shared carbocationic intermediate 34 into either
5-epi-aristolochene (12) or premnaspirodiene (13).
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the farnesyl cation, experiments with a fluorinated substrate
analogue indicated that the formation of the germacryl cation
was indeed a concerted process due to optimal overlap of the
p-orbital of the C2,C3 double bond with the C1–O bond facili-
tated by Tyr 92 (together with i.a. Phe 112 and Phe 178) (vide
infra).29
In studies on the bacterial enzyme pentalenene (16) synthase
(PS), the roles of the active site residues Phe 76 and Phe 77 were
examined by separate replacement with alanine, resulting in
massive reductions of the catalytic activity in both cases.
Anomalous germacrene A production by these mutants might
have been considered to be due to alternate cation stabilisation
but instead was proposed, on energetic grounds, to be a derail-
ment of the cyclisation rather than perturbation of the stabilities
of carbocations by altered amino acid residues.16 Alteration of
Mg2+ binding motifs in FS-TS has also been observed to lead to
increased promiscuity in the product spectrum.20,28
EIZS has been the subject of SDM experiments, both of the
Mg2+ binding motifs44 and the aromatic portions of the active
site.23 In the former case, as with other sesquiterpene synthases,
altered products were formed, presumably due to altered coor-
dination geometries of the metal ions at the active site cleft.44
Alteration of the active site aromatic residues Phe 95, Phe 96 and
Phe 198 resulted in alternate products but the formation of these
could not be assigned purely to either perturbation of cation–p
stabilisation or a change of the active site contour and the role of
these residues remains ambiguous.23
TEAS and Hysocyamus premnaspirodiene (13) synthase
(HPS) are two evolutionarily related enzymes that share
a common carbocationic intermediate but catalyse different
rearrangements of this cation (Scheme 2). In an impressive
demonstration of artificial control of sesquiterpene synthase
activity, various amino acid residues in spheres of increasing
radii around the active site contours of these two enzymes were
identified and changed reciprocally to those of the other enzyme.
Thereby each enzyme was converted to the activity of the other,
indicating that close control over substrate folding and structural
dynamics are crucial to product specificity and demonstrating the
potential for modification of these enzyme activities for bespoke
synthetic biology purposes. The enzyme is clearly not an unin-
volved spectator as amino acid residues in and around the active
sites of TEAS and HPS determine which of two alkyl groups
migrates from a common carbcationic intermediate.45 It should,
however, be noted here that for several sesquiterpene synthases
from plants like amorpha-4,11-diene (18)46 or d-cadinene (22)
synthases (V. Gonzalez and R. K. Allemann, unpublished
results), such an exchange of activities through the replacement
of residues in or around the active sites were unsuccessful, in that
only a reduction of the activities were observed. Saturation
mutagenesis of GA-DCS using error-prone PCRwas only able to
produce a small number of clones that produced germacrene
D-4-ol instead of d-cadinene (22).47
Another successful example of man-made control of sesqui-
terpene cyclisation is the manipulation of the highly promiscuous
GHS from Abies grandis (52 known products34) towards much
higher fidelity production of some of its minor products; after
determination of residues in the two spheres around the active
site of GHS a computer algorithm was used to predict the
mutations required to alter the activity of GHS in the desired
64 | Nat. Prod. Rep., 2012, 29, 60–71
directions. With only five mutation GHS was converted from
a promiscuous sesquiterpene synthase into a variety of highly
active enzymes with reduced product spectra (Scheme 3).48
Recently a group of sesquiterpene synthases from the fungus
Coprinus cinereus have been examined in detail.49,50 Cop6
produces predominantly (�)-a-cuprenene (42). Cop4 is much
more promiscuous, with (+)-d-cadinene (22), b-copaene (43) and
b-cubebene (44) as major products, while Cop3 makes predom-
inantly a-muurolene (45) and (+)-germacrene A (32) (Scheme 4).
The binding pockets of Cop3 and Cop4 (as judged by homology
models based on the crystal structure of AT-AS) seem to be much
larger than that of Cop6, which might explain the greater
promiscuity. A SDM study looked at the exchange of the H1-
a loop between these sesquiterpene synthases, mirroring
a previous study with an a-farnesene synthase from an apple
species.51 The H1-a loop closes over the active site upon binding,
moving the NSE motif into the correct position for catalysis.
Various mutants altered the ability of the loop to close over the
active site leading to premature quenching of the cations and
hence production of germacrenes.
An interesting case of the flexibility of sesquiterpene synthase
catalysed chemistry has been provided by studies of the biosyn-
thesis of the trisnor-sesquiterpene geosmin (50). Although not
strictly a sesquiterpene, this compound is formed by a sesquiter-
pene synthase that eliminates acetone from an intermediate ses-
quiterpenoid alcohol to generate its product (Scheme 5).
Geosmin synthase is a bifunctional enzyme from Streptomyces
coelicolor52 that possesses functionally different C-and N-
terminal catalytic domains with significant sequence similarity to
pentalenene synthase. Cleavage of the protein into the two
separate domains gave two functional enzymes; the N-terminal
half was found to convert FDP to germacradienol (48) and
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(�)-germacrene D (25),52 while the C-terminal half is responsible
for the conversion of germacradienol to geosmin (50).53 A series
of mutant geosmin synthases with changed DDXXD and NSE
Mg2+ binding motifs provided insight into the mechanism; most
notably the S233A mutant produced wild-type products together
with 11% of isolepidozene (47), a neutral putative cyclopropyl
intermediate that is attacked by water under acid catalysis to give
germacradienol prior to conversion to geosmin.53
3.2 Aza-analogues
An alternative approach to SDM for the study of cation stabi-
lisation and conformational control in sesquiterpene synthase
catalysis is the use of synthetic analogues of the proposed
cationic intermediates. Nitrogen containing compounds that are
either quaternised or simply protonated in aqueous solution to
generate a cation, have been used in kinetic studies and as
structural analogues for single crystal X-ray analyses (for
examples of aza analogues see Fig. 2).
Scheme 3 Site directed mutagenesis of g-humulene synthase fromG. aroreum
the above sesquiterpene products.
Scheme 4 The major products generated by sesquiterpene synthases
Cop3 and Cop6 from Coprinus cinereus.
Fig. 2 Aza-analogues of sesquiterpenoid cationic intermediates.
This journal is ª The Royal Society of Chemistry 2012
In the crystal structure of wild-type EIZS, BTAC (51) was
observed to orient itself with aromatic residues.23 The crystal
structure of EIZS-F198A coordinated to three Mg2+ ions, PPi
and BTAC revealed a different orientation of the cation relative
to the wild type structure, implying that the resulting expansion
of the active site volume allowed other orientations of cation
binding and hence altered product specificity.23
A recent crystal structure of FS-TS-Y305F in complex with the
potent inhibitor 53 indicated that this aza-analogue bound to the
enzyme in a conformation that did not mimic the proposed
orientation of a-bisabolyl cation (6) during the catalytic
(GHS) has been used to alter the enzyme to a specific synthase for each of
Nat. Prod. Rep., 2012, 29, 60–71 | 65
Scheme 5 The mechanism for the biosynthesis of geosmin (50).
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cycle.17,54 Favourable interactions with PPi seemed to cause the
observed binding orientation rather than cation–p interactions
with aromatic residues, suggesting that sesquiterpene synthases
enforce cation location and formation through kinetic rather
than thermodynamic control.17
Use of BTAC in crystal structure analysis of mutant FS-TS
provided the first definitive proof that a cation can bind in the
active site of a sesquiterpene synthase since aza-bisabolyl cations
were of unknown protonation state in other known structures.22
An apparent ‘loosening’ of the coordination of PPi and Mg2+ in
FS-TS structures led to the interpretation that this leads to
greater versatility of PPi to act as a general base (vide infra) and
for the observed diversity of products for this enzyme.
Recently, aza-analogues designed to resemble eudesmane
cation have been prepared and tested as inhibitors of PR-AS. In
contrast to the observations discussed above for FS-TS, one of
these compounds (54, Fig. 2) proved to be a potent inhibitor of
PR-AS in both the presence and absence of PPi. This suggested
that this compound may be a genuine mimic of eudesmane cation
since no obvious favourable interactions with PPi were detec-
ted.55 The trigonal aza-analogues 55 and 56, designed to mimic
the likely transition state between (�)-germacrene A (10) and
eudesmane cation (33), inhibit PR-AS synergistically with PPi,
implying a role for PPi in carbocation formation, perhaps as the
active site acid.56
Scheme 6 Mechanisms for the biosynthesis of amorpha-4,11-diene (18).
3.3 Substrate analogues
Many of the finer points in the mechanism of formation of inter
alia (+)-aristolochene (11),30,57 trichodiene (20),32,58 d-cadinene
(22),59,60 epi-cubenol (24),61–64 pentalenene (16),65–67 and (�) and
(+)-germacrene D (25 and 26)68 were uncovered using deuter-
ated, tritiated and carbon-14 labelled analogues of FDP and
NDP. Much of this work has been reviewed previously.3,4,11
More recently, the catalytic mechanism of amorpha-4,11-diene
synthase from Artemisia annua (ADS) has been studied using
labelled FDP analogues. In contrast to the antimalarial
compound artemisinin, which is synthesised from amorpha-4,11-
diene, ADS has not been studied extensively from a mechanistic
point of view. Two parallel studies with (R)- and (S)-[1-2H]-FDP
66 | Nat. Prod. Rep., 2012, 29, 60–71
(1b and 1c) established a 1,3-shift of HSi on C1 of FDP to C7
implying a mechanism that proceeds via a-bisabolyl cation (6),
which is formed by 1,6 ring closure of NDP (Scheme 6).69,70
Interestingly, deuterium incorporation was observed upon
incubation in >75% 2H2O. Although the position of incorpora-
tion was not determined, the authors speculated that an inter-
mediate cation may be quenched and reprotonated as a side
reaction from the main catalytic pathway.70 This may hint at the
alternate reaction pathways to some of the minor products that
have been observed for this enzyme and present opportunities for
engineering altered reactivities into this enzyme.71 It will be
interesting to see how the enzyme promotes 1,6 ring closure of cis
farnesyl cation rather than the 1,10 ring closure catalysed by
cadalane producing sesquiterpene synthases.
Further to the SDM studies on the related enzymes TEAS and
HPS, deuterium labelling experiments were used to analyse the
stereochemistry and timing of protonation, rearrangement and
elimination steps for these two enzymes.72 Incubation with both
enantiomers of [8-2H]-FDP (1d and 1e), tris- and hexa-deuterated
FDP analogues at C12 and C13 (1f, 1g and 1h) and incubation of
unlabelled FDP in 2H2O gave results that were broadly in line
with mechanistic observations for the fungal aristolochene syn-
thases,57,72,73 namely that the proton lost upon formation of the
intermediate germacrene A is from C12; the intermediate is then
reprotonated from the Re face and HSi is lost from C8 to form
the final product.
A new trend in sesquiterpene synthase research is the hunt for
new activities using genome mining. Two presumptive sesqui-
terpene synthase genes from Streptomyces clavuligerus were
recently identified and codon optimised synthetic genes con-
structed and overexpressed in E. coli. The gene products were
identified as related sesquiterpene synthases producing the
cadalane family sesquiterpenoids (-)-d-cadinene (22) and (+)-T-
muurolol (23).74 Incubation of the enzymes with 1b and 1c and
analysis of the products by 1H and 2H NMR spectroscopy
revealed a [1,3]-hydride shift of HSi on C1 of FDP to C11 in the
reactions catalysed by both enzymes. This is in line with a similar
stereochemical prediction for the hydride shift observed for GA-
DCS.59 Further to this, a promiscuous cadalane producing
sesquiterpene synthase MtTPS5 from Medicago truncatula has
recently been identified.35 Incubations of unlabelled FDP in2H2O showed C15 proton/deuteron exchange, which was pre-
dicted through an alternative mechanism for cadalane produc-
tion that was first postulated many years ago but until this report
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had no experimental support.75 Some of the products appeared
to be generated via germacrene D, which after protonation at
C15 reacts to a variety of bicyclic products. Interestingly, the
alteration of GA-DCS by saturation mutagenesis led to an
enzyme that produced a close analogue of germacrene D,47
implying that only subtle differences in enzyme structure and
dynamics differentiate between production of germacrene D or
germacrene D-4-ol and d-cadinene from a common intermediate.
Scheme 7 Biosynthesis of epi-isozizaene (17).
Scheme 8 The biosynthesis of avermitilol (66). Virifloridol (67), ger-
macrene A (32) and germacrene B (63) are minor byproducts.
Fig. 3 Deuterated substrate analogues discussed in the text.
Another enzyme that produces germacrene D as a by-product
is geosmin synthase. Interrogation of the mechanism of this
enzyme using 1b and 1c and analysis of the products by GC-MS
established that HSi on C1 of FDP is the species that undergoes
[1,3]-hydride transfer in this case.76 Despite producing the same
enantiomer of germacrene D, (-)-GDS from Solidago canadensis
proceeds via [1,3]-migration of HRe on C1 of FDP indicating that
the two enzyme must convert FDP to (-)-germacrene D (25)
through different catalytic mechanisms.68,76 The mechanism of
the dominant geosmin production was established to result from
retro-Prins fragmentation of germacradienol by labelling studies.
Incubation of unlabelled FDP in 2H2O resulted in specific
incorporation of several deuterium atoms in geosmin and the
by-products although not in the (-)-germacrene D.77 The use of
tris-deuterated FDP at C13 (1h) resulted in trapped acetone
containing the label. This confirmed the retro-Prins fragmenta-
tion of 47 and the use of 1j as a substrate and analysis of the
resulting product demonstrated a predicted [1,2]-hydride shift
from C2 of FDP.78
Genome mining studies first identified EIZS from Strepto-
myces coelicolor. Incubation with 1b and 1c, [1-2H2]-FDP (1j)
and the trisdeutero-FDP analogues 1g and 1h established the
current understanding of the mechanism of this reaction
including the stereochemistry of the methyl migration in the
penultimate step (Scheme 7).44,79
Another genome mining study, this time of Streptomyces
avermitilis, identified a gene encoding a previously undiscovered
sesquiterpene synthase that produces the eponymous avermitilol
(66).80 The stereochemistry of the ring closures was elucidated
using 1b and 1c. The enzyme also produces small amounts of
(+)-germacrene A (32), germacrene B (63) and viridiflorol (67)
(Scheme 8).80
The product of presilphiperfolan-8b-ol (72) synthase from
Botrytis cinerea is a common precursor in the biosynthesis of
This journal is ª The Royal Society of Chemistry 2012
a series of toxins produced by this necrotic plant pathogen.81
With the use of deutero-FDP analogues it was established that
the reaction sequence proceeds via humulyl cation (5) and does
not require the intermediacy of NDP (3) (Scheme 9), although
this compound is an alternate substrate of this enzyme.
Fluorinated isoprenyl diphosphates have been used in the past
to study the mechanism of enzymes involved in terpene biosyn-
thesis,82–84 but only recently have they been extensively employed
for the analysis of sesquiterpene synthases. Their behaviour as
either inhibitors or substrates has revealed several subtle mech-
anistic details that may not otherwise have come to light. The
crystal structures of AT-AS and GA-DCS have now been solved
in the presence of several fluorinated sesquiterpene analogues
(vide supra).14,21 Fig. 4 illustrates the fluorinated FDP analogues
that have been used in this work and also shows several of the
fluorinated sesquiterpenoids produced with various sesquiter-
pene synthases.
Nat. Prod. Rep., 2012, 29, 60–71 | 67
Scheme 9 Mechanism for the biosynthesis of presilphiperfolan-8b-ol
(72).
Fig. 4 Fluorinated FDP analogues referred to in the text and some of
the germacrene A analogues resulting from turnover of these analogues
by sesquiterpene synthases.
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Fluorinated FDP analogues have been used to interrogate the
mechanism of the PR-AS; 12,13-FF-FDP (1n) was instrumental
in showing the role of Phe 112 (and other bulky active site resi-
dues) in aligning the distal double bond for attack at C1 during
PRAS catalysis (vide supra).29 In addition, 2F-FDP (1k),31 6F-
FDP(1l)85 and 14F-FDP (1o)85 have proved to be efficient
substrates of PR-AS, which converts these fluorinated FDPs to
analogues of (�)-germacrene A (73–75, Fig. 4). This provides
further evidence of the intermediacy of germacrene A in the
production of eremophilenyl sesquiterpenes such as (+)-aristo-
lochene (11) since the presence of fluorine on C2 or C6 prevents
the cyclisation of (�)-germacrene A by reduction of electron
68 | Nat. Prod. Rep., 2012, 29, 60–71
density on the C2,C3 or C6,C7 double bonds, respectively, and
also by preventing the build up of positive charge on C3 in the
eudesmane cation (34). In the case of fluorine on C14, there is
again a depletion of electron density in the C6,C7-double bond,
which reduces the p-basicity and so prevents protonation of 10.
It also shows that these enzymes can be utilised to produce
analogues of natural products that may be exploitable in the
future for medicinal or agrochemical uses. 6F-FDP (1l) is also an
efficient substrate of TEAS, giving the corresponding fluorinated
(+)-germacrene A analogue (76) as the sole product.86
The inclusion of TEAS amongst the so-called high-fidelity
sesquiterpene synthases has recently been challenged by the
observation of several minor products in its product pool
originating from both transoid and cisoid ring closures of
FDP.87 The amount of cisoid products increased dramatically
when the enzyme was incubated with the 2Z isomer of FDP.88
In addition, crystals of TEAS and a promiscuous mutant
(M4 TEAS) were soaked with 2F-FDP and 2-fluoro-2E-FDP to
provide snapshots of the binding modes of substrates in
conformations leading to both of these cyclisation pathways.89
Comparison of the X-ray structures showed that both substrates
were bound in the active site with the farnesyl chain in
a U-shaped conformation but with differing orientations
(inversions) of the first two isoprene units – hence showing
complete occupancy of the active site and also displaying
conformations consistent with the expected orientation that
would lead to their observed product pools. The cisoid substrate
analogue, however, was not bound in an active conformation
since the C–O bond was coplanar with the C2,C3 bond, in
contrast to the transoid substrate where the C–O bond was
perpendicular to this and hence oriented optimally for the
reaction. These results show that while the enzyme active site
surface contour is important for the control of product
outcome, it appears that an interplay exists between enzymatic
enforcement of substrate conformation and the intrinsic
conformational possibilities of the substrate; the use of a cisoid
C2,C3 double bond can produce dramatically altered products.
Interestingly, it was also observed that several of the mutations
in the M4 mutant TEAS cause little change to the contour of
the active site. Since this mutant is much more promiscuous
than the wild-type enzyme, this hints strongly at dynamic effects
playing an important part in the product outcome for this
enzyme. The electron density of the farnesyl chain in the crystal
structures of this mutant soaked with the two substrate
analogues showed discontinuities, perhaps indicating increased
motional flexibility of the substrate in the active site relative to
the wild type enzyme; this is fully consistent with the promis-
cuity observed for this mutant when incubated with FDP.
In agreement with results revealing active site plasticity in
GHS,48 the active site contour of PR-AS has been shown to be
highly plastic for binding FDP analogues. Three phenyl-FDP
analogues are potent competitive inhibitors of this enzyme.90
This reveals potential for synthetic biology applications, since it
may be possible to engineer a larger active site cavity to allow
productive binding of such bulky substrate analogues. It also
reveals that the active sites of these enzymes are not static and
rigid cavities but are able to adopt different shapes to accom-
modate bulkier ligands hinting at the dynamic role played by
these proteins during catalysis.
This journal is ª The Royal Society of Chemistry 2012
Scheme 10 The mechanism of formation of (S)-b-bisabolene (77) and
(S)-b-macrocarpene (19) catalysed by TPS6 and TPS11 from maize.
Scheme 11 Patchouli alcohol (80) and two of the minor hydrocarbon
products, b-patchoulene (81) and a-guaiane (82) produced by patchouli
alcohol synthase from Pogostemon cablin.
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4 Acid/base catalysis
Many sesquiterpene synthases require participation of acids and
bases during their catalytic cycle. For example, the two enan-
tiomers of germacrene A are thought to be neutral intermediates
in the pathways to (+)-aristolochene (11) (via 10) and 5-epi-
aristolochene (via 32) requiring an acid to regenerate a cation;
a base is then required in the final step of the reaction.11 Various
candidates have been proposed to act as active site acids/bases
including amino acid side chains, active site water molecules as
well as the diphosphate generated from the substrate itself.
Based on the proposal that Tyr 520 may be the active site acid
responsible for protonation of germacrene A in catalysis by
TEAS,91 it was suggested that the analogous function is fulfilled
by Tyr 92 in the active site of PR-AS.27 However, since
replacements of Tyr 92 in PR-AS lead to enzymes that still
produce significant quantities of (+)-aristolochene (11), Tyr 92
cannot be the active site acid.40 PPi has since been suggested to
act as the acid/base for AT-AS on the basis of crystallographic
analysis but there remains little other than structural evidence for
this.19 Meanwhile, a theoretical study proposed that instead of
being a reaction intermediate, (�)-germacrene A (10) could be
a by-product of PR-AS produced via a different catalytic
mechanism to the major product.92 Instead, it was proposed
based on gas-phase calculations that there may be a direct
hydride transfer between C12 and C6, or a proton transfer via an
intermediary water molecule. Such a mechanism would bypass
the neutral germacrene A and avoid an energetically expensive
sequence of cation quenching followed by cation regeneration.92
This attractive mechanistic proposal had to be rejected, however,
since in deuterium labelling experiments the stereochemistry of
protonation at C6 was inconsistent with either direct hydride
transfer or transfer by way of a water molecule.73 This labelling
study established that the protonation of 10 occurs on the same
face of the molecule as the later deprotonation event that
produces the final product, implying that the same species is
involved in both reaction steps.73 A similar study on TEAS
showed that protonation occurs on the same face of (+)-germa-
crene A in TEAS catalysis.72
The idea of tyrosine as an active-site acid in TEAS has proved
enticing for other sesquiterpene synthases also. Y295 has been
proposed as the active site base responsible for final deprotona-
tion in FS-TS catalysis – however, SDM of this residue ruled out
this possibility, since mutants had similar activity to the wild type
enzyme. Once more the PPi group most likely acts as the proton
donor/acceptor.20
Further enzymes where tyrosine has been investigated as an
active site acid are the (S)-b-macrocarpene (19) synthases TPS6
and TPS11 frommaize, which possess a tyrosine residue (Tyr 522
in both) that appears to be an evolutionarily conserved residue
(cf. Tyr 520 in TEAS).33 (S)-b-Macrocarpene was shown by
deuterium labelling studies to arise from protonation of a neutral
intermediate ((S)-b-bisabolene, 77) and SDM of Y522 sup-
pressed this protonation activity yielding b-bisabolene as
product (Scheme 10). Additionally, protonation of (S)-b-bisa-
bolene was found to be bothMg2+ and pH dependent, as has also
been reported for TEAS protonation of (+)-germacrene A (32).93
A similar pH sensitivity has been observed to affect the product
profile of Cop4 from Coprinus cinereus. This, however, appears
This journal is ª The Royal Society of Chemistry 2012
to be a protein folding effect since SDM experiments where H235
and N239 were altered to proline and leucine, respectively,
resulted in abolition of the pH dependence of product
formation - probably due to hydrogen bonding changes within
the enzyme.49 Whatever the specific identity of active site acids/
bases in various sesquiterpene synthases, it is clear that amino
acid residues are at least in part responsible for such processes in
some cases – notably TEAS and in TPS6 and TPS 11 from maize
and that the enzyme thereby plays a crucial active chemical role
in the catalysis.
Finally, a recent interrogation of patchouli alcohol (80,
Scheme 11) synthase from Pogostemon cablin using the singly
labelled [2-2H]-FDP 1j showed a surprising result. In addition to
the expected singly labelled patchouli alcohol product (�65% of
isolated alcoholic material), a doubly deuterated product was
also observed (�35% of isolated alcohols).94 From the positions
of deuteration it was concluded that an intermediate alkene
structure becomes protonated (or deuterated when 1j is used as
substrate) to give the final product. Minor hydrocarbon by-
products b-patchoulene (81) and a-guaiene (82) were depleted of
deuterium, indicating the source of the second deuteron.
Comparison of the amino acid sequence with other sesquiterpene
synthases and use of an active site model identified Leu 410 as an
active site residue that may undergo reorientation during the
catalytic cycle, effectively creating a second pocket in the active
site were the observed deuteration events occur.94 This may be
another example of active enzyme participation in catalysis.
Nat. Prod. Rep., 2012, 29, 60–71 | 69
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5 Conclusions
All of the work described here has helped unravel the general
principles that control the mode of action of sesquiterpene
synthases. Even though not the subject of this review, it is worth
noting that the same principles will also operate during catalysis
by mono- and diterpene synthases. Despite a lack of significant
sequence homology between sesquiterpene synthases in plants,
insects, bacteria and fungi, the chemistry that they facilitate
within the shared class I terpene synthase fold is the basis for
the generation of the enormous diversity in structure and
stereochemistry found in natural sesquiterpenes. The plasticity
of the sesquiterpene synthases appears to provide a framework
for the combinatorial production of many natural terpenoids
though subtle alterations in the composition of the active site
during evolution. The enzymes bind their substrates in reactive
conformations that take advantage of the chemical reactivity
inherent in FDP. This first binding step is central for the initi-
ation of the Mg2+-dependent expulsion of diphosphate and the
product profile. Perhaps not surprisingly, there is complexity in
the formation of the Michaelis complex of PR-AS21 and perhaps
all sesquiterpene synthases, in that after the initial molecular
recognition of FDP, coordination to Mg2+B reorients the
substrate diphosphate group. Subsequently, binding of Mg2+Ctriggers a more substantive conformational change leading to
active site closure. Finally, binding of Mg2+A completes the
formation of the trinuclear metal ion cluster and triggers further
structural changes to complete active site closure and to
generate the cyclisation-competent conformation of FDP. In the
Michaelis complex the three negative charges of the PPi leaving
group are fully neutralized to facilitate ionization and initiation
of the cyclisation cascade. It is likely that diphosphate stays
bound throughout the reaction thereby providing stability to
the carbocationic transition states and intermediates. The parts
of the active site that bind the isoprenoid tail of the substrate
are largely made up of aliphatic and aromatic amino acids to
allow additional stabilisation of the carbocations of the reaction
cascades through cation–p or charge–quadrupole interactions.
The specific orientation of the active site residues allows the
enzymes to channel the reaction along a specific pathway to
generate the desired outcome and at the same time strictly
controls water access.
Finally, general acid and base catalysis is involved in
terpene synthase reactions. There has so far been little
evidence that the enzymes provide amino acids to act as acids
and bases and, by exclusion, it is now assumed that in many
cases the bound diphosphate ion helps move protons. The
selectivities of the protonation and deprotonation reactions
depend on the correct orientation of acid and bases with in the
active sites.
Sesquiterpene synthases play an active controlling role in
catalysis; they provide a fascinating example of how enzymes
have evolved to take advantage of the chemical properties
inherent in their substrates to generate a biologically desirable
outcome with remarkable efficiency and specificity. (Sesqui)-
terpene synthases, the chemistry they facilitate, their biology
and their applications will no doubt continue to astound us
through a combination of structural complexity and chemical
simplicity.
70 | Nat. Prod. Rep., 2012, 29, 60–71
6 Acknowledgments
We are grateful for the financial support of the Biotechnology
and Biological Sciences Research Council (grants 6/B17177; BB/
G003572/1 and BB/H01683X/1), the Engineering and Physical
Sciences Research Council (grant EP/D06958/1) and Cardiff
University. We thank Dr Juan A. Faraldos, Dr Sabrina Touchet,
DrMahmoud Akhtar, Dr RobertMart and Dr E. Joel Loveridge
for critical comments on the manuscript and past and present
members of the terpene cyclase group for stimulating
discussions.
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