Structural elucidation of cisoid and transoid cyclization pathways of a sesquiterpene synthase using 2-fluorofarnesyl diphosphates

Article abstract of DOI:10.1021/cb900295g

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP (1b) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (cis,trans)-FPP (1b) to exclusively (?99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-trans FPP (1a) and (cis,trans)-FPP (1b) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed.

Full text of DOI:10.1021/cb900295g

ARTICLE
Structural Elucidation of Cisoid and Transoid
Cyclization Pathways of a Sesquiterpene
Synthase Using 2-Fluorofarnesyl Diphosphates
Joseph P. Noel†,‡, Nikki Dellas‡,§, Juan A. Faraldosʈ,#, Marylin Zhaoʈ, B. Andes Hess, Jr.Ќ,
Lidia SmentekЌ,¶, Robert M. Coatesʈ, and Paul E. O’Maille†,‡,؉,
*
†Howard Hughes Medical Institute, ‡The Salk Institute for Biological Studies, Jack H. Skirball Center for Chemical Biology &
Proteomics, 10010 Torrey Pines Road, La Jolla, California 92037, §Department of Chemistry, University of California San
Diego, 9500 Gilman Drive, La Jolla, California 92093, ʈDepartment of Chemistry, University of Illinois at Urbana–
Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, ЌDepartment of Chemistry, Vanderbilt University,
Nashville, Tennessee 37235, and ¶Institute of Physics, Nicolaus Copernicus University, 87-100 Torun´, Poland. #Present
address: Cardiff School of Chemistry, Cardiff University, Cardiff, U.K. ϩPresent address: Department of Metabolic Biology,
John Innes Centre (JIC), Phytochemicals and Health, Institute of Food Research (IFR), Colney Lane, Norwich NR4 7UH, U.K.
erpenes comprise the most structurally diverse
class of natural products, playing essential eco-
logical roles by mediating communication be-
tween plants and insects, by providing antimicrobial de-
fenses for plants, and likely acting in additional
ABSTRACT Sesquiterpene skeletal complexity in nature originates from the
enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and
subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation path-
ways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, pro-
duces cisoid products as a component of its minor product spectrum. To investi-
gate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP
(1b) as an alternative substrate. Strikingly, TEAS was catalytically robust in the en-
zymatic conversion of (cis,trans)-FPP (1b) to exclusively (Ն99.5%) cisoid prod-
ucts. Further, crystallographic characterization of wild-type TEAS and a catalyti-
cally promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-trans FPP
(1a) and (cis,trans)-FPP (1b) revealed binding modes consistent with preorganiza-
tion of the farnesyl chain. These results provide a structural glimpse into both ci-
soid and transoid cyclization pathways efficiently templated by a single enzyme ac-
tive site, consistent with the recently elucidated stereochemistry of the cisoid
products. Further, computational studies using density functional theory calcula-
tions reveal concerted, highly asynchronous cyclization pathways leading to the
major cisoid cyclization products. The implications of these discoveries for ex-
panded sesquiterpene diversity in nature are discussed.
T
undefined capacities (as reviewed previously (1)). Terpe-
noids originate from primary isoprenoid metabolism,
wherein iterative condensation of 5-carbon isoprene
units (isopentyl diphosphate and dimethylallyl diphos-
phate) catalyzed by prenyltransferases produce poly-
isoprenoid diphosphate substrates of varying lengths
(for a review, see ref 2). Terpene synthases, in turn, of-
ten referred to as cyclases given the cyclic nature of
many of their products, transform the polyisoprenoid
diphosphate substrates, e.g., geranyl diphosphate C10,
farnesyl diphosphate C15, or geranylgeranyl diphos-
phate C20, into structurally diverse mono-, sesqui-, and
diterpene products, respectively.
The structural complexity of these molecules under-
lies their diverse biological activities. Ruzicka formu-
lated the biogenetic isoprene rule, which predicted the
formation of sesquiterpenes arising from the head-to-
tail connection of three isoprene units (5 carbons each)
where the skeletal complexity can be formally deduced
from farnesol (3). Cane later developed a general stereo-
chemical model for sesquiterpene biogenesis involving
the idealized fold of the farnesyl chain in the active site
posing the reacting carbons to direct a sequence of elec-
trophilic cyclizations and rearrangements following py-
*Corresponding author,
paul.o’maille@bbsrc.ac.uk
Received for review November 23, 2009
and accepted February 22, 2010.
Published online February 22, 2010
10.1021/cb900295g
© 2010 American Chemical Society
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rophosphate loss/ionization (4). Moreover, a limited
number of conformations of the farnesyl substrate give
rise to much greater product diversity. Product specific-
ity or in many cases product diversity arises from a lim-
ited number of farnesyl chain conformations, wherein
the reacting double bonds reside mutually perpendicu-
initial C1 attack on the distal C10ϪC11 double bond,
prior to further downstream cyclizations (Figure 1). By
contrast, the “cisoid” synthases conduct an initial
C2ϪC3 double bond isomerization prior to cyclization,
wherein the nascent farnesyl cation is recaptured at C3
by pyrophosphate to form the neutral, enzyme-bound
lar to a common plane. Thus, there is a direct correspon- (3R)- or (3S)-nerolidyl diphosphate (NPP), thus allowing
dence between the absolute stereochemical configura-
tion of the sesquiterpene product and the inferred
conformation of the precursor (4). A central challenge
for the structural enzymologist is to define how indi-
vidual terpene synthases statically or dynamically dis-
criminate between alternative polyprenyl cation confor-
mational modes or selectively favor particular
rotation about the C2ϪC3 bond from trans to cis. Reion-
ization of NPP generates the 2,3-cisoid farnesyl cation,
which undergoes further cyclization via initial C1 attack
either on the proximal C6ϪC7 or on the distal C10ϪC11
double bonds prior to further transformations. This reac-
tion mechanism has been invoked to account for the for-
mation of ␤-macrocarpene (8), amorpha-4,11-diene (9),
conformations to shepherd reactive intermediates along trichodiene (10), and cedranes such as isocedrol (11).
distinct cyclization cascades.
Structural biology provides a framework for address-
ing the evolutionary origins of complex terpenoid me-
Moreover, the biosynthesis of epi-isozizaene was re-
cently described in connection with the isolation and
functional characterization of epi-isozizaene synthase
tabolites and their biosynthetic pathways. Terpene syn- from Streptomyces coelicolor (12). The cisoid mechanis-
thases comprise a structurally conserved enzyme family, tic class in sesquiterpene cyclases is akin to the major-
which adopt a common ␣-helical architecture termed
ity of monoterpene cyclases that proceed via the cis
the class I terpenoid cyclase fold, first revealed from the (neryl) allylic cation to form the corresponding cyclic
crystal structures of tobacco 5-epi-aristolochene syn-
thase (TEAS) from Nicotiana tobaccum (5) and pentalene
monoterpene products (for a review, see ref 13).
Though most sesquiterpene synthases can be classi-
synthase from Streptomyces UC5319 (6). The lyase func- fied as belonging to either the cisoid or transoid classes,
tion of these enzymes stems from two conserved metal some display cryptic activities associated with the other
binding motifs: the “aspartate-rich” DDxxD motif that co- pathway. Notably, TEAS catalyzes the formation of (ϩ)-
ordinates two Mg2ϩ ions and the “NSE/DTE” motif that
coordinates a third Mg2ϩ. These static X-ray crystallo-
5-epi-aristolochene (1), the first committed step in the
biosynthesis of the phytoalexin capsidiol, the principal
graphic studies show that the binding of (Mg2ϩ)3-PPi sta- component of tobacco’s antifungal chemical defense
bilizes the active site in a closed conformation that is se- (14). Aside from its major product, TEAS generates an
questered from bulk solvent (7). In addition to multiple
divalent cation coordination bonds, the PPi anion ac-
cepts hydrogen bonds from conserved basic residues
additional 24 minor products, some of which are de-
rived from the cisoid cyclization pathway (15). The struc-
tural variations of these cisoid products result from a
when bound to the closed synthase conformation, while multistep mechanism of cyclizations and rearrange-
a hydrophobic pocket, lined by a number of aromatic ments, suggesting that TEAS templates the cisoid cat-
residues, cradles the farnesyl chain and most likely tem- ion pathway with fidelity and enables the formation of
plates the cyclization reaction by enforcing particular
substrate conformations and stabilizing carbocations
through ␲-stacking interactions.
a distinct set of complex skeletal structures. These unex-
pected observations give rise to several confounding
questions. Does a single parental fold of the farnesyl
chain give rise to products along both the cisoid and
While the wealth of structural diversity among ter-
pene hydrocarbons arises from bifurcations along multi- transoid pathways in TEAS? On a structural level, how
step cyclization pathways, divergence at the earliest
mechanistic step defines two major classes of terpene
synthases and hence distinct product families. The
“transoid” synthases ionize (trans,trans)-farnesyl
diphosphate (FPP) (1a) to generate the 2,3-transoid far-
are both pathways templated within a single active site?
How can the cryptic cisoid pathway in TEAS become ac-
tivated? Does this “vestigial” activity portend an unan-
ticipated new function for TEAS in tobacco?
To address these questions, we investigated the ci-
nesyl cation (trans along the C2ϪC3 bond) followed by soid cyclization recently discovered in TEAS using syn-
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TABLE 1. Enzymatic products from incubations of TEAS wild-type and the M4 mutant with (cis,trans)-
or (trans,trans)-FPP
% by mass
TEAS wt
% by total ion count (TIC)
Peak
Product
RT (min)
TEAS wt
TEAS M4
(cis,trans)-FPP products
3
2
4
5
6
7
8
9
10
(Ϫ)-␣-cedrene
(ϩ)-2-epi-prezizaene
␣-acoradiene
4-epi-␣-acoradiene
(Ϫ)-␤-curcumene
nerolidol
␣-bisabolol
epi-␣-bisabolol
cis-farnesol
15.3
15.8
16
16.1
16.5
17.1
18.6
18.6
18.8
n.a.
18.95
18.85
46.52
6.80
2.54
12.30
0.90
0.11
0.11
0.29
11.58
18.48
30.54
13.40
4.06
4.65
1.81
0.05
0.05
0.50
26.46
37.89
3.44
1.03
13.78
3.61
1.80
1.80
5.69
11.99
remaining
(trans,trans)-FPP products
11
1
12
13
germacrene A
15.1
16.2
16.3
16.48
n.a.
3.65
78.90
6.21
1.66
9.58
10.98
30.66
27.46
25.47
5.43
5-epi-aristolochene
4-epi-eremophilene
premnaspirodiene
remaining
thetically derived (cis,trans)-FPP (1b), a geometrical iso- the predicted stereochemical course of the reaction. Fur-
mer of the native all-trans substrate (Figure 1, panel a). ther, key transition state geometries calculated using
[The descriptors “cis” and “trans” in (cis,trans)-FPP and density functional theory revealed concerted, highly
the fluoroFPP isomers refer to the longest carbon chain asynchronous cyclization pathways. Thus, combining
about the 2,3 and 6,7 double bonds, respectively, as
defined in Figure 1, panel a. For more formalized no-
menclature, see refs 16 and 17. ] Remarkably, TEAS
efficiently converts this alternative substrate into pre-
dominantly (ϩ)-2-epi-prezizaene (2), a novel sesqui-
terpene hydrocarbon related to the naturally occurring
alcohol jinkohol (18), along with other cisoid cation-
derived products. Large-scale enzyme reactions pro-
duced sufficient amounts of hydrocarbon products for
stereochemical elucidation and positive identification of
nine compounds (18).
biochemical, computational, and crystallographic analy-
ses with the recently elucidated stereochemistry of the
cisoid products, we pictorially reconstruct herein the
TEAS-catalyzed transoid and cisoid cyclization path-
ways. Further, comparison of wild-type and mutant
TEAS-analogue complexes provides structural snap-
shots and insights into product specificity/diversity re-
flected in the preorganization of the farnesyl chain along
2 major cyclization pathways.
RESULTS AND DISCUSSION
In the present investigation, crystallographic analy-
TEAS-Directed Cisoid Cyclization with
ses of wild-type TEAS and a previously reported promis- (cis,trans)-FPP. To investigate the cryptic cyclization ac-
cuous mutant (TEAS M4) (19), with unreactive 2-fluoro
analogues (2a and 2b) of (trans,trans)- and (cis,trans)-
FPP (1a and 1b, respectively) revealed catalytically rel-
tivity in TEAS, we synthesized the 2,3-cis geometrical
isomer of farnesyl diphosphate (cis,trans)-FPP (1b) (18).
This substrate analogue is effectively “preisomerized”,
evant binding modes and distinct farnesyl chain topolo- and hence its ionization by TEAS would be expected to
gies that are consistent with preorganization by the
active-site for cisoid or transoid cyclization, and hence,
generate the cisoid farnesyl cation, which in turn should
feed directly into the cisoid cyclization pathway
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Figure 1. Mechanism of TEAS-catalyzed cyclization of (cis,trans)-FPP to (؉)-2-epi-prezizaene (2). a) The structure and semisys-
tematic nomenclature for isomers of the FPP substrate and fluorinated analogues used in this study are indicated. b) Based on
biochemical and stereochemical information governing the nature of the cisoid products, a cyclization mechanism is proposed
to account for all identified products along this multistep pathway (18). c) The configuration of the terminal isopropenyl tail of
the (7R)-␤-bisabolyl cation relates to the final cyclization products of the cisoid pathway.
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(Figure 1, panel b). Indeed, our pilot experiments re-
vealed that TEAS generated a near exclusive spectrum
of cisoid products when incubated with (cis,trans)-FPP
(1b) as substrate, including the previously reported iso-
prezizaene ((ϩ)-2-epi-prezizaene, 2) as the dominant re-
action product (Table 1 and Figure 2). This result demon-
strated the ability of TEAS to template the cisoid
cyclization pathway with a high degree of product speci-
ficity and catalytic efficiency.
As detailed in a concurrent report, the structure, ster-
eochemistry, and enantio-purity was determined for
nine cisoid products of TEAS isolated from large-scale
enzyme incubations with (cis,trans)-FPP (1b), an
achievement enabling the formulation of a mechanistic
proposal for their biosynthetic origin (Figure 1, panel b)
(18). Chromatographic separations or enrichments of
five hydrocarbon and three alcohol fractions, together
with comparative NMR spectral data, chiral GC analyses,
optical rotation measurements, and chemical correla-
tions, allowed assignment of structures for (ϩ)-2-epi-
prezizaene (2), (Ϫ)-␣-cedrene (3), ␣-acoradiene (4),
4-epi-␣-acoradiene (5), (Ϫ)-␤-curcumene (6), nerolidol
(7), the ␣-bisabalol epimers (8 and 9), and 2,3-cis-
farnesol (10) shown in Figure 1, panel b and listed in
Table 1. Importantly, knowledge of the relative and ab-
solute stereochemistry of the cisoid hydrocarbon prod-
ucts provided vital information for guiding computa-
tional studies and accurately describing the
stereochemical course of the TEAS cisoid cyclization re-
action mechanism (Figure 1, panel b and below).
To further characterize the product specificity of wild-
type TEAS and a previously described promiscuous mu-
tant (A274T V372I Y406L V516I) referred to here as M4
TEAS (19), we performed GCϪMS analyses via the vial
assay (20) to examine product mass spectra derived
from native (1a) and (cis,trans)-FPP (1b) substrates
(Figure 2 and Table 1). TEAS generated 18 distinct ter-
pene products from the 2-cis substrate, including (ϩ)-2-
epi-prezizaene (2), which constitutes nearly half the
product spectrum (46% by TIC). With the native all-trans
FPP (1a) substrate, M4 TEAS exhibits relaxed product
specificity in the terminal steps of the transoid cycliza-
tion pathway, producing roughly equal amounts of
5-epi-aristolochene (1), 4-epi-eremophilene (12), and
premnaspirodiene (13). With (cis,trans)-FPP (1b), M4
TEAS produced the same repertoire of hydrocarbons as
Figure 2. Gas chromatograms of products from incubations of
wild-type TEAS and the M4 mutant with (cis,trans)- and
(trans,trans)-FPP. TEAS or its M4 mutant were incubated with
either (a) (trans,trans)-FPP (1a) or (b) (cis,trans)-FPP (1b) using the
vial assay followed with analysis by GC–MS as described in
Methods. Major product peaks are labeled according to identified
products listed in Table 1.
(ϩ)-2-epi-prezizaene (2). This result indicates that the
catalytic promiscuity of M4 TEAS extends to the cisoid
pathway.
To assess the efficiency of the enzymatic conversion
of (cis,trans)-FPP (1b) to (ϩ)-2-epi-prezizaene (2) by
wild-type TEAS and M4 TEAS, we conducted steady-
state kinetic experiments (Table 2). The experiments us-
ing (trans,trans)-FPP (1a) reveal that both enzymes dis-
play comparable catalytic efficiencies (kcat/Km), with the
higher Km of M4 TEAS (13.3 ␮M) offset by a higher over-
all turnover number (9.4 minϪ1). Both enzymes utilize
(cis,trans)-FPP (1b) with similar catalytic efficiencies,
with the promiscuous TEAS M4 possessing a Km
the wild-type enzyme, again displaying relaxed product (7.9 ␮M) lower than that of wild-type but also a slower
specificity with only a third of the turnovers producing
overall turnover (4.6 minϪ1), the reverse of the trend ob-
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Figure 3. Computational analysis of the TEAS cisoid cyclization pathway. a) Density functional theory (DFT) calcula-
tions were performed on the TEAS cisoid pathway and revealed concerted, highly asynchronous reactions with a
transition state (14 or 15) specific to the formation of (؊)-␣-cedrene (3) or (؉)-2-epi-prezizaene (2), respectively
(red arrows). Hong and Tantillo (26) previously located an alternative concerted highly asynchronous transition in
the cedrene pathway (blue arrow). b) A transition state structure (14) was discovered that connects the (6S)-␣-
bisabolyl cation to the (4S)-␣-acorenyl cation corresponding to point 50 on the intrinsic reaction coordinate plot.
The migrating hydrogen (dark blue) and the two carbons (yellow) forming a nascent ␴-bond (dashed line) are de-
picted. The plot shows the change in the dashed bond distance (ࡗ), the original (9) and new (Œ) C؊H bond dis-
tances of the migrating hydrogen in 14 during the course of the reaction taken from an IRC calculation. The transi-
tion state structure is point 0. c) A transition state structure (15) along the (؉)-2-epi-prezizaene (2) pathway is
shown. Change in bond distances shown during the course of the IRC calculations bond (ࡗ, a bond; , b bond; Œ,
c bond) indicates the highly asynchrous nature of this step. The transition state structure (15) is point 10.
served for (trans,trans)-FPP (1a). Additionally, enzy-
matic activities were assessed using the 2-fluoro ana-
logues of (trans,trans)-FPP (2a) and (cis,trans)-FPP (2b)
(Figure 1, panel a). Wild-type TEAS and several of its
catalytically active mutants fail to turn over either (2-
trans,6-trans)-2-fluorofarnesyl diphosphate (trans-2F-
FPP, 2a) or (2-cis,6-trans)-2-fluorofarnesyl diphosphate
(cis-2F-FPP, 2b) after 24-h incubation periods. The lack
of measurable catalytic activity when using the fluoro-
substituted FPPs is most likely due to a strong electron-
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withdrawing inductive effect due to the presence of the
spectively. Immediately following the initial C1ϪC6 cy-
fluoro substituent at position 2 that prevents ionization clization to the ␣-bisabolyl cation, water quenching
and pyrophosphate loss. By contrast, the fungal sesqui- again intercepts the cyclization path by indiscriminant
terpene cyclase aristolochene synthase converted trans- attack on either face of the cation to produce equal
2F-FPP (2a) cleanly to 2-fluorogermacrene A after ex-
tended incubation times (21), and trichodiene synthase ing approximately one-third of the alcohol products. Al-
produced several fluorinated sesquiterpene hydrocar- ternative proton eliminations from C5 of the (7R)-␤-
bons of unknown structure (22). Although unreactive flu- bisabolyl cation account for the third most abundant
amounts of ␣- and epi-␣-bisabolol (8 and 9), compris-
oro analogues have been useful for crystallography
with limonene synthase (23) and more recently 2F-FPP
complexes with aristolochene synthase (24), the inert-
product in the TEAS cisoid spectrum of products,
namely, (Ϫ)-␤-curcumene (6), representing 16% of to-
tal hydrocarbon product. Finally, ␣- and 4-epi-␣-
ness of the 2-fluoro-FPPs proved useful for our crystallo- acoradienes (4 and 5) stem from proton elimination
graphic experiments.
from the terminal isopropenyl tail of the acorenyl cat-
ions, representing the remaining products observed at
4% and 1.2% total hydrocarbon, respectively.
Computational Analysis of the TEAS Cisoid
Stereochemical Mechanism of Cyclization. On the
basis of the elucidated stereochemistry of the major ci-
soid products, a reaction mechanism for the TEAS-
catalyzed cyclization of the cisoid farnesyl cation is pro- Mechanism. The intrinsic reactivity, conformation, and
posed (Figure 1, panel b) (18). Catalysis begins with energy of carbocation intermediates define physically al-
divalent cation-assisted ionization of (cis,trans)-FPP gen- lowable cyclization pathways, which ultimately pass
erating the cisoid farnesyl cation. The ensuing 1,6 cy-
clization involves C1 attack on the re face of C6 of the
C6ϪC7 double bond to produce the (6S)-␣-bisabolyl
cation. This step is followed by a 120° CW rotation and
through the selectivity filter of active site geometry and
electrostatics, most likely modulated by enzyme dynam-
ics. To computationally examine the conformation and
intrinsic energetics of the cisoid cyclization pathway, we
6,7 hydride shift to form the (7R)-␤-bisabolyl cation. The conducted density functional theory (DFT) calculations.
(7R)-␤-bisabolyl cation is a key reaction intermediate, ly- While numerous transition states were identified which
ing at the intersection of the majority of cisoid hydrocar- connect consecutive intermediates in the proposed re-
bon products. The orientation of the terminal isoprene
unit at this stage directs the subsequent divergence of
reaction trajectories at the C6ϪC10 cyclization step
(Figure 1, panel c). When the isoprene unit is oriented
action mechanism (Figure 1), alternative connectivities
were discovered that bypass adjacent carbocations,
thereby directly linking more distal steps in the cisoid cy-
clization pathway (Figure 3, panel a). For example, a
endo, the C6ϪC10 cyclization produces the (1R,4R,5S)- transition structure (14) was found that bypasses the
␣-acorenyl cation. This intermediate undergoes a further (7R)-␤-bisabolyl cation in a concerted, highly asynchro-
C3–C11 cyclization and then a Wagner–Meerwein re-
arrangement to a tertiary carbocation prior to proton
elimination to produce (ϩ)-2-epi-prezizaene (2). Con-
nous reaction by directly connecting the (6S)-␣-bisabolyl
cation to the (4S)-␣-acorenyl cation along the pathway
to (Ϫ)-␣-cedrene. Following this, a transition structure
versely, the exo configuration leads to the (1R 4S,5S)-␣- was found for the next step, linking the (4S)-␣-acorenyl
acorenyl cation, possessing the opposite stereochemis- cation to the final carbocation, thereby completing this
try at C4 relative to the aforementioned prezizaene
pathway. A C2–C11 cyclization of this cation followed
by proton elimination terminates the reaction pathway
at (Ϫ)-␣-cedrene (3).
The remaining stereochemically defined products
comprise roughly equal amounts of sesquiterpene hy-
drocarbons and alcohols, and their formation can be ra-
tionalized as branches off the main reaction pathway
(Figure 1, panel b). Early in the mechanism, water
quenching on C1 or C3 of the nascent cis-farnesyl cat-
ion accounts for cis-farnesol (10) and nerolidol (7), re-
pathway (via 14) from the (6S)-␣-bisabolyl cation to (Ϫ)-
␣-cedrene (3) (Figure 3, panel b). Interestingly, the (6S)-
␣-bisabolyl cation to the (4R)-␣-acorenyl connection was
not uncovered due to steric occlusion, indicating the im-
portance of the (7R)-␤-bisabolyl cation along the path-
way to (ϩ)-2-epi-prezizaene (2).
Formally, the formation of (ϩ)-2-epi-prezizaene (2)
involves a high-energy secondary carbocation from
the anti-Markovnikov C3ϪC11 cyclization. It has been
suggested that such high-energy secondary carboca-
tions in terpene biosynthesis can be avoided in the
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TABLE 2. Kinetic parameters of TEAS wild-type and the M4 enzyme determined using either (cis,trans)- or
(trans,trans)-FPP
(trans,trans)-FPP
KM (␮M)
(cis,trans)-FPP
KM (␮M)
kcat (min؊1
)
kcat/KM (␮M؊1 min؊1
)
kcat (min؊1
)
kcat/KM (␮M؊1 min؊1
)
TEAS wt
TEAS M4
2.5 Ϯ 0.7
9.43 Ϯ 0.21
8.4 Ϯ 0.89
13.31 Ϯ 1.04
0.3
0.71
5.71 Ϯ 0.08
4.62 Ϯ 0.07
14.03 Ϯ 2.59
7.97 Ϯ 1.26
0.41
0.58
gas phase via concerted, highly asynchronous mecha- sis (5). Several residues were excised from both the
nisms, in analogy to the formation of the C and D
rings in the cyclization of squalene oxide to lano-
wild-type and M4 TEAS models during refinement due
to a lack of clearly observable electron density and the
sterol (25). These mechanisms have also been discov- attendant poor refinement of these regions (Supplemen-
ered in related computational studies of sesquiter-
pene cyclization. This was indeed the case here as
we located a transition structure (15) and demon-
tary Figure S2). As previously noted, the mutations in
the M4 TEAS protein reside either in the active site
(V516I) or distribute more peripherally around the ac-
strated with intrinsic reaction coordinate (IRC) calcula- tive site surface (A274T, V372I, and Y406L) with dis-
tions that it connected the distal cyclization events
leading to (ϩ)-2-epi-prezizaene (2) in a concerted,
highly asynchronous step (Figure 3, panel c). Hong
and Tantillo have independently identified this same
transition state (26).
tances from the active site center ranging from 7 to 14
Å (Supplementary Figure S3). While each mutated side
chain was readily discernible in the electron density, no
significant backbone distortions were evident, strongly
hinting at dynamic, not static, modulation of the active
site contour for templating transformations of farnesyl
cations in TEAS. However, the V516I mutation directly af-
Structure of Wild-Type TEAS and M4 TEAS with 2-
Fluoro Analogues. We expected that the three-
dimensional structures of TEAS-2F-FPP complexes would fects the active site contour with implications for sub-
be informative regarding the static templating of both ci- strate binding as discussed below.
soid and transoid pathways in the TEAS active site. To
investigate the structural basis for substrate preorgani-
zation and catalytic promiscuity along the transoid and
Observable electron density is present in the active
site regions for all complexes, and the positions of
ligand-binding residues were clearly established with
cisoid cyclization pathways, we carried out crystal soaks the exception of Y527 on the JϪK loop. In all the struc-
with the nonionizable substrate analogues trans-2F- tures, contiguous electron density stretches from the
FPP (2a) and cis-2F-FPP (2b), respectively. These experi- DDxxD motif through the diphosphate moiety into the
ments yielded proteinϪsmall molecule complexes dif-
fracting to resolutions ranging from 2.1 to 2.6 Å
(Table 3).
NSE/DTE motif enshrouding the catalytically essential
Mg2ϩ ions (Figure 4, panel c). Although three Mg2ϩ ions
are visible in each complex, a complete octahedral coor-
Global comparison of all structures by superposition- dination sphere of waters is only discernible in the high-
ing C-␣ carbons revealed a high degree of similarity with est resolution M4 TEAS-cis-2F-FPP complex. Electron
root mean square deviation (rmsd) values ranging from
0.22 to 0.37 Å for all atoms (Supplementary Table S1).
Annotating structures according to B-factors suggested
a common pattern of dynamic regions across all struc-
tures refined (Supplementary Figure S1). In contrast to
density surrounding the diphosphate appendage is the
most prominent feature in the calculated electron den-
sity (without ligands modeled) with large ␴ values in the
SIGMAA-weighted 2Fo Ϫ Fc electron density maps
(Figure 4, panel b). Clear electron density extends from
the originally published TEAS·farnesyl hydroxyphospho- the diphosphate through the first isoprene unit contain-
nate (FHP) structure, all complexes described here ex- ing the fluoro substituent in all complexes but trails off
hibit disorder in a portion of the JϪK catalytic loop, a re- through the center of the chain and picks up again at the
gion encompassing amino acids 521Ϫ533 that distal isoprene unit (Figure 4, panel e). Despite the wan-
completes the enclosure of the active site during cataly- ing electron density for the distal isoprene units, the far-
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Figure 4. Crystallographic analysis of wild-type and M4 TEASs bound to fluoro-FPPs. a) Global structure of TEAS is illustrated as a
rainbow-colored ribbon with the active site region boxed. b) Zoomed-in view of the Mg2؉-diphosphate coordination complex of the
M4 TEAS-cis-2F-FPP complex with the 2Fo ؊ Fc map contoured at 3␴. c) Close-up view of the DDxxD motif (residues 301, 302 (not
shown), and 305), neighboring NSE/DTE motif (residues 444, 448, and 452), coordinating Mg2؉ and diphosphate in the indicated
fluoro-farnesyl diphosphate complexes contoured at 1␴ in the 2Fo ؊ Fc SIGMAA-weighted electron density map. d) Close-up of the
TEAS-cis-2F-FPP complex active site showing the bound ligand and the neighboring TEAS residues. e) Ligand density for the respec-
tive complexes with the SIGMAA-weighted 2Fo ؊ Fc electron density map contoured to either 1␴ (dark blue) or 0.6␴ (light blue).
nesyl chain clearly curls into a U-shape in all com-
plexes, particularly at lower ␴ where continuous density required for maximum activity, the CϪO bond adopts a
is apparent in the wild-type complexes (Figure 4, parallel position in the cis-2F-FPP structures and hence
panel e). Taken together, these complexes display near represents an inactive conformation. If this conforma-
pendicular to the plane of the C2ϪC3 double bond as
complete occupancy (based upon the unmistakable
diphosphate and first isoprene unit electron density),
tion were reflective of the (cis,trans)-FPP binding, then
rotation of the C1ϪC2 bond would be required to form
and aside from an incomplete JϪK loop, Mg2ϩ ions and a catalytically active complex. This inactive conforma-
ligands are bound with the farnesyl chain folded in a
manner consistent with the formation of major cycliza-
tion may be promoted by the 2-fluoro moieties in cis-2F-
FPP (2b) through its electrostatic interaction with Arg
tion products along both the transoid and cisoid mecha- 264 residing 3 Å away (Figure 4, panel d).
nistic pathways.
Spatial Reconstruction of Cisoid and Transoid
Comparison of ligand binding modes between the
Reaction Pathways in TEAS. Multiple substrate binding
cis- and trans-2F-FPP complexes reveals important differ- modes discerned during building and refinement for the
ences relating to catalysis. While the orientation of the extended farnesyl chain could potentially satisfy, and
CϪO bond in both trans-2F-FPP structures is nearly per- likely contribute to, the observed electron densities
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Figure 5. Spatial reconstruction of the transoid and cisoid cyclization pathways in TEAS. a) Refined conforma-
tions for the trans-2F-FPP or cis-2F-FPP·TEAS complexes are displayed in the binding pocket (clipped surface),
and models of indicated reaction intermediates or products were manually positioned relative to the refined
conformations. An accompanying schematic of chemical structures designates the 2-fluoro positions in each
substrate as H(F). Images were rendered with UCSF Chimera (57). Transition state structures are shown along-
side their corresponding rendered figures; dashed lines are used to indicate bond breakage and formation.
b) Proposed substrate folds leading to the cisoid and transoid cyclization pathways in TEAS.
(Supplementary Figure S4). Despite these ambiguities,
the general topology of the farnesyl chain is clear and
consistent with the anticipated parental fold inferred
from the elucidated stereochemistry of the final prod-
ucts. Importantly, electron density for the cis-2F-FPP
complexes reveals that the terminal isoprene unit curls
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a
TABLE 3. Crystallographic data and refinement statistics
wt TEAS-trans-2F-FPP
wt TEAS-cis-2F-FPP
M4 TEAS-trans-2F-FPP
M4 TEAS-cis-2F-FPP
pdb code
3M01
3M0
3LZ9
3M00
Space group
P41212
P41212
P41212
P41212
Unit-cell parameters:
a(Å)
125.5
125.5
126.3
126.1
b(Å)
125.5
125.5
126.3
126.1
c(Å)
122.7
121.3
121.9
122.4
␣-␤-␥°
90
90
90
90
Monomers per Asymm unit
Resolution range (Å)
No. reflections measured
Merging R-factor
͗I/␴͘
Completeness
Redundancy
No. reflections used
R-factor
1
1
1
1
500.0Ϫ2.6
219537
0.093 (0.325)
17.28 (4.63)
0.963 (0.914)
7.36 (7.48)
30227
0.2065
0.2423
547
500.0Ϫ2.5
324161
0.077 (0.282)
23.53 (5.12)
0.979 (0.938)
8.54 (8.55)
30047
0.1976
0.2257
547
500.0Ϫ2.28
377780
0.090 (0.346)
16.04 (3.59)
0.992 (0.903)
7.74 (6.53)
22460
0.1935
0.2464
547
500.0Ϫ2.1
248702
0.080 (0.358)
16.45 (3.55)
0.956 (0.872)
4.02 (3.92)
57483
0.2205
0.2586
547
Free R-factor
No. amino acid residues
No. water molecules
150
168
174
412
aValues in parentheses represent highest resolution shell.
into a helical (endo) fold in accordance with the antici-
pated conformation (Figure 1, panel c). This orients the
tion of the substrate must occur to produce the com-
pact (ϩ)-2-epi-prezizaene (2) final product. The crucial
plane of the C10–C11 double bond parallel to a poten- elements of preorganization are the juxtaposition of C1
tially attacking carbocation at C1. In contrast, the plane and C6 together with the endo orientation of the farne-
of the C10–C11 double bond of the terminal isoprene
syl tail, both consistent with the observed electron den-
unit is perpendicular to a nascent C1 carbocation in the sity of the TEAS·cis-2F-FPP complex. Therefore, the static
trans-2F-FPP complexes, in accord with an initial C1–
C10 cyclization along the transoid pathway.
To spatially reconstruct the two major cyclization
picture drawn from these observations reveals a catalyti-
cally relevant substrate binding conformation and sub-
strate/intermediate preorganization very early along the
pathways in TEAS, we manually docked transition state cisoid pathway catalyzed by TEAS. Based on this model,
structures and models of the major products into the re- the pyrophosphate ion would reside close by but suit-
spective active sites of wild-type trans-2F-FPP and cis-2F- ably sequestered by neighboring interactions to stabi-
FPP complexes (Figure 5, panel a). Restraining the place- lize the developing positive charge of the secondary car-
ment of products/intermediates such that cyclization to bocation while limiting recapture probability prior to
a specific product most likely proceeds with the mini-
the final proton elimination yielding (ϩ)-2-epi-
mal amount of conformational distortion for the nascent prezizaene (2).
farnesyl cation en route to the final products results in
For the transoid pathway, the key transition state
a mechanistically plausible transition state geometry en structure leading to the major product (ϩ)-5-epi-
route to the observed dominant product.
aristolochene (1) involves a methyl migration atop the
Superposition of the transition state structure (15)
decalin ring system of the eremophilyl carbocation
on the farnesyl chain indicates that substantial contrac- (Figure 5, panel a, right) as previously reported (19). To
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achieve this energetically favorable alkyl migration, sub- tant offer a glimpse into the structural underpinnings of
strate folding must preorganize an initial electrophilic at- product specificity or lack thereof. To discern the struc-
tack of C1 on C10 of the distal double bond. This re-
quires substantial movement of the chain following
ionization, as these atoms are 5 Å apart in the ground
state complexes. However, judging by the degree of
tural basis for product specificity in both cisoid and tran-
soid cyclization pathways, we conducted a compara-
tive analysis of wild-type TEAS and M4 mutant structures
with particular attention focused on the active site con-
overlap between the transition state model and the far- tour and farnesyl chain binding modes. The most obvi-
nesyl chain, this motion can be accommodated largely ous surface distortion, whether statically or dynamically
within the first isoprene unit with minimal conforma-
tional adjustments of the more distal isoprene units.
derived, is contributed by the V516I mutation, which in-
troduces a methyl group into the active site cavity
Therefore, this conformation of the farnesyl chain is con- (Supplementary Figure S5). While no drastic distortion
sistent with TEAS preorganizing FPP (or more likely the is evident in the comparative models of the farnesyl
resultant acyclic farnesyl cation) for cyclization to 5-epi- chain, the electron density for the farnesyl chain in the
aristolochene (1), in contrast to the original TEAS·FHP M4 TEAS structures is discontinuous, indicative of in-
complex (5). The phosphate moiety of FHP overlaps with creased dynamic motion and/or local disorder (Figure 4,
the ␤-phosphates of 2F-FPPs, although the farnesyl
chain is more extended and folds in essentially the op-
posite direction (Supplementary Figure S4).
Cisoid Cyclase Activities with (trans,trans)-FPP. On
the basis of our spatial reconstruction of the cisoid cy-
clization pathway, we propose a model describing the
panel e). Interestingly, only the M4 TEAS-cis-2F-FPP com-
plex exhibits a significant shift in the position of Y520,
which additionally alters the active site surface features.
The ligand-dependency for the Y520 shift may reflect
the interaction between the farnesyl chain, wherein the
central isoprene unit is inverted relative to the corre-
“cisoid fold” of (trans,trans)-FPP (1a) that is representa- sponding wild type complex, and active site residues in
tive of the preorganization of the farnesyl chain leading defining the preorganized binding state. Despite the
to its conversion to cisoid-cation-derived hydrocarbons. higher resolution of the M4 structures, the density for
Accordingly, an alternative, catalytically productive bind- the ligand is discontinuous, even when the electron
ing mode of FPP is populated in which the farnesyl
chain curls into its typical U-shaped topology, but with
the first two isoprenoid units inverted relative to the
density maps are viewed at low ␴, in contrast to the wild-
type structures (Figure 4, panel e). It is probable that a
more dynamic farnesyl chain in M4-TEAS-cis/trans-2F-
“transoid fold” configuration (Figure 5, panel b). The ci- FPP structures explains the lack of electron density for
soid binding mode therefore possesses the DU configu- the entire farnesyl chain consistent with this M4 TEAS’s
ration, opposite to that described for germacrene A
(27), and importantly, with the distal isoprenoid unit
curled below this plane into an alternative binding
pocket formed by T401, T402, C440, R441, D444, and
the diphosphate moiety (Figure 4, panel a). We posit
that the positioning and anchoring of the terminal iso-
prenoid unit is an essential stereochemical feature for
triggering ionization, perhaps through both steric and
promiscuous catalytic activity along both pathways.
Conclusions. (cis,trans)-FPP proved effective in direct-
ing reactions along the cisoid cyclization pathway in TEAS.
The isolation and stereochemical elucidation of the prod-
ucts lead to formulation of reasonable reaction path-
ways to the cisoid-derived sesquiterpene skeletons (18).
Traditionally, chemical tools have been a vital part of de-
fining the stereochemical course of terpene biosynthesis,
electronic effects. Upon ionization, a kinetically slow ini- as elegantly exemplified by the use of (1R)-[1-3H]- and
tial isomerization occurs as the C10–C11 double bond
(1S)-[1-3H]-geranyl diphosphate to provide direct experi-
is rotated out of position for electrophilic attack by C1; in mental confirmation that cyclization along the cisoid
turn, a re face capture by the pyrophosphate ion on C3 pathway results in net retention of configuration at C1 of
of the nascent farnesyl cation generates the neutral (3S)- the substrate (28). Fluoro isoprenoid diphosphate sub-
NPP intermediate. Rotation around the C2–C3 bond fol- strate analogues also have been instrumental in elu-
lowed by reionization generates the 2,3-cis-farnesyl cat- cidating mechanistic aspects of terpene biosynthesis,
ion and entry into the cisoid cyclization pathway.
Structural Picture of Catalytic Promiscuity. The
ligand–protein structures of a promiscuous TEAS mu-
most notably through the interception of reaction inter-
mediates such as 6-fluorogermacrene A (29) and
7-fluoroverticillenes (30) in TEAS and taxadiene synthase
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ARTICLE
enzymes, respectively. Most recently, 2F-FPP and 12,13-
difluorofarnesyl diphosphate (DF-FPP) were instrumental
in deciphering the probable order of metal-ion binding
diphosphate captured another instance where the iso-
prenoid chain conformation is consistent with the geom-
etry of the final product (23). However, it has been noted
and conformational changes required for catalysis by aris- that most reported crystal structures of terpene syn-
tolochene synthase from Aspergillus terrus (24).
To date, crystallographic analyses of terpene cy-
clases have yielded important insights into how these
thases complexed with isoprenoid substrate analogues,
including 2F-FPP used here, reveal isoprenoid tail con-
formations that are not catalytically relevant (5, 23, 24,
enzymes function on the atomic scale. Most notably, an- 31). Considering the ideal case, where there is unam-
choring the diphosphate moiety of the substrate and
metal coordination by the DDxxD and NSE/DTE motifs
biguous density for every atom of the farnesyl chain, a
central challenge in the field remains to resolve struc-
shown in crystal structures paints a picture of the funda- tural features responsible for product specificity or lack
mental role of these events in terpene synthase cataly- thereof from a static picture alone, given the degeneracy
sis. Structures containing inorganic pyrophosphate or a of possible products arising from a single parental sub-
substrate analogue bound in the active site display or-
dering of various loops proximal to the active site, con-
sistent with a closed protein conformation that shields
reactive carbocation intermediates from solvent (5, 23,
31). Further, alterations in pyrophosphate binding are
strate fold. Future progress toward defining the origins of
sesquiterpene skeletal complexity will undoubtedly
benefit from integrating dynamic information from NMR
and time-resolved fluorescence (in progress) with com-
putational approaches and protein crystallography to
thought to aid in the modulation of prenyl chain orienta- develop a much clearer and time-resolved biophysical
tion within the active site and most likely modulate the
fate of the early intermediates along prescribed mecha-
nistic pathways (32). All structures reported in the cur-
picture of terpene synthase directed cyclization.
What possible relevance does the cryptic cisoid cy-
clization pathway of TEAS have in the natural world? Al-
rent study contain the full complement of Mg2ϩ ions co- though (cis,trans)-FPP has not been identified as a me-
ordinating the diphosphate of the fluoro-farnesyl
analogues. However, despite this clear coordination
geometry, elements of the JϪK loop remain disordered
in both wild-type and mutant structures bound to
tabolite in tobacco or related Solanaceous plants, a
(cis,trans)-farnesyl diphosphate synthase has been
identified in Mycobacterium tuberculosis involved in
bacterial cell wall synthesis (33, 34), suggesting the po-
diphosphate containing ligands with Y527 electron den- tential relevance of this compound in other biological
sity missing from the active site. This lack of observ-
able density for Y527 stands in contrast to the original
systems. Moreover, while often observed, the biologi-
cal significance of small amounts (3–14% of total prod-
TEAS·FHP complex where this residue is clearly discern- uct) of (cis,trans)-FPP formation by FPP synthases (35)
ible (Supplementary Figure S2).
has been ignored to date. Is it possible then that TEAS
possesses a “moonlighting” role in vivo by gathering up
what we would normally consider biosynthetic “waste”
and recycling it into a bioactive product? While TEAS pro-
duces cisoid terpenes in vitro, the presence of these me-
These observations hint at a greater role of dynam-
ics in terpene chain cyclization than evident from the
early structural work based upon static crystal struc-
tures. The wild-type ligand complexes in the current
study revealed density for the farnesyl chain folded in a tabolites has yet to be confirmed in planta. Nonethe-
manner consistent with catalysis, and this can be inter- less, TEAS clearly possesses an efficient catalytic
preted in light of the established stereochemistry for all potential to access presently unanticipated in vivo
TEAS products. Moreover, these static observations en-
abled the positing of 2 distinct parental folds each of
which gives rise to either cisoid or transoid cyclization
pathways for TEAS (Figure 5, panel b). The structure of
a complex of limonene synthase with 2-fluorolinalyl
chemical diversity from lengthy branches of the cisoid
reaction pathway, a property that may have been natu-
rally selected for and that can also be immediately ex-
ploited for biotechnological applications starting with
(cis,trans)-FPP.
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Protein expression, purification, and kinetic measurement
are detailed in Supporting Information.
METHODS
Organic Synthesis. (cis,trans)-FPP was available from the
concurrent investigation (18). (trans, trans)- and (cis,trans)-
2-FluoroFPPs were accessed from the corresponding 2-
fluorofarnesol isomers (30) by conversion to the respective
2-fluorofarnesyl chlorides and SN2 displacements with (nBu)4N/
diphosphate (HOPP) (36) with complete retention of the 2,3-
double bond configurations by means of procedures similar to
those reported previously (see Supporting Information) (30).
(cis,trans)-2-FluoroFPP has not been previously described in the
literature. Characterization data for ammonium salt of 2b are
as follows: white solid (51 mg, 68%); 1H NMR (CD3OD, 400 MHz)
␦ 5.17–5.11 (m, 1H, vinyl H), 5.11–5.05 (m, 1H, vinyl H), 4.59
(dd, 2H, J ϭ 23.3, 5.4 Hz, CH2OPP), 2.16–2.11 (m, 4H, CH2),
2.09–2.04 (m, 2H, CH2), 2.00–1.95 (m, 2H, CH2), 1.68 (d, 3H,
J ϭ 3.5 Hz, CH3), 1.66 (q, 3H, J ϭ 1.2 Hz, CH3), 1.61 (d, 3H, J ϭ
1.2 Hz, CH3), 1.60 (br d, 3H, J ϭ 0.6 Hz, CH3); 31P NMR (CD3OD,
162 MHz) ␦ Ϫ7.99 (br d, J ϭ 14.9 Hz), Ϫ9.30 (br d, J ϭ 14.1 Hz);
19F NMR (CD3OD, 376 MHz) ␦ Ϫ118.9 (td, J ϭ 23.2, 3.5 Hz).
Protein Crystallization and Data Collection. Crystallization of
purified proteins was conducted using hanging drops over a
0.5-mL reservoir (15% w/v PEG8K, 200 mM Mg(OAc)2, 100 mM
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO)-Naϩ,
pH 7.0). Crystal soaks were conducted overnight in reservoir so-
lution containing 10 mM fluoro-farnesyl diphosphate ligand.
Crystals were frozen in soak solution also containing 20% v/v
ethylene glycol as cryoprotectant. Data collection was performed
at the Stanford Synchrotron Radiation Laboratory (SSRL) beam-
line 1–5 for wild-type TEAS-2F-FPP and cis-2F-FPP complexes,
while data for the M4 TEAS mutant structures were collected at
the Advanced Light Source (ALS) beamline 8.2.1. All data were
processed using XDS software (37). The initial crystallographic
structure solutions were obtained through molecular replace-
ment analyses using the TEAS-FHP complex (PDB id 5eat) as the
search model with Molrep in Collaborative Computational
Project No. 4 (CCP4) (38). Model building was performed using
COOT (39) and rounds of refinement were conducted using Crys-
tallography NMR System (CNS) (40). To refine the position of
the farnesyl chain, the first isoprene unit containing the 2-flouro
group was built and refined, followed by sequential addition,
building and refinement of remaining isoprene units. For the
wild type cis-2F-FPP complex, additional multi-conformer refine-
ment was undertaken using the program RefMac (41–48) in
CCP4 (38). The fold inferred from the known stereochemistry of
the final products was built into the density followed by a final
round of refinement using CNS to produce the current models of
the farnesyl chain (Supplementary Figure S5).
Computational Methods. All calculations were performed
with GAUSSIAN 98W and GAUSSIAN 09W (49) and the density
functional method using B3LYP, Becke’s three-parameter hybrid
method (50) with the Lee–Yang–Parr correlation functional (51)
and the 6-31G* basis set (52). All stationary points were con-
firmed with second derivative calculations. Energies reported
here include zero-point energy corrections calculated with un-
scaled B3LYP/6-31G* frequencies obtained analytically with
G98W. Intrinsic reaction coordinate calculations (53, 54) were
used to determine reaction pathways. Single point mpw1pw91/
6-311ϩG(2d,p)//B3LYP/6-31G* calculations as recommended
by Matsuda (55) were carried out for all stationary points re-
ported (56).
Product Elucidation. A preparative-scale incubation was car-
ried out using 127 mg (310 mmol) of (cis,trans)-FPP and a total
of 18 mg of recombinant wild-type TEAS in order to accumulate
sufficient material for chromatographic fractionations, NMR
analyses, and optical rotation measurements of the major prod-
ucts as described (18).
Acknowledgment: L.S. acknowledges financial support from
the Polish Ministry of Science and Education (research project
no. N N 202 187636) This work was supported by the Howard
Hughes Medical Institute and the National Science Foundation
Grant MCB-0645794 (J.P.N.).
Supporting Information Available: This material is available
free of charge via the Internet at http://pubs.acs.org.
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