Mechanism of action and inhibition of dehydrosqualene synthase

Article abstract of DOI:10.1073/pnas.1010907107

"Head-to-head" terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg²⁺ cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

Full text of DOI:10.1073/pnas.1010907107

Mechanism of action and inhibition of
dehydrosqualene synthase
a,1
b,c,d,1
a
e
e
c,d
c,d
e
Fu-Yang Lin , Chia-I Liu
, Yi-Liang Liu , Yonghui Zhang , Ke Wang , Wen-Yih Jeng , Tzu-Ping Ko , Rong Cao ,
a,e,2
b,c,d,2
Andrew H.-J. Wang
, and Eric Oldfield
a
b
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; Institute of Biochemical Sciences,
c
d
National Taiwan University, Taipei 106, Taiwan; Institute of Biological Chemistry and Core Facility for Protein Production and X-ray Structural Analysis,
Academia Sinica, Taipei 115, Taiwan; and Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801
e
Edited* by Frances H. Arnold, California Institute of Technology, Pasadena, CA, and approved October 11, 2010 (received for review July 26, 2010)
“
Head-to-head” terpene synthases catalyze the first committed
converted by the same enzyme to dehydrosqualene, which is
further transformed to STX (8). Because STX is a so-called
virulence factor for S. aureus (9), inhibiting its formation is of
interest in the context of developing new routes to antiinfective
therapies (10).
steps in sterol and carotenoid biosynthesis: the condensation of
two isoprenoid diphosphates to form cyclopropylcarbinyl diphos-
phates, followed by ring opening. Here, we report the structures of
Staphylococcus aureus dehydrosqualene synthase (CrtM) com-
plexed with its reaction intermediate, presqualene diphosphate
Given the key role of the head-to-head tri- and tetraterpene
synthases in sterol and carotenoid biosynthesis, there has been
remarkably little work reported on their three-dimensional struc-
tures. There has been one report of the structure of human SQS
with a bound inhibitor (11), but relatively little mechanistic infor-
mation was obtained because the inhibitor was not obviously sub-
strate, intermediate, or product-like. In our group, we reported
the X-ray crystallographic structure of CrtM from S. aureus (10).
There were two substrate-analog inhibitor binding sites (sites
S1 and S2), but determining which represented the prenyl donor
(the “allylic” FPP that ionizes to form the 1′ carbocation) and
which represented the prenyl acceptor (that provides the C2,3
alkene group) was not attempted because the 1′-2,3 distances for
both possible assignments were ∼5 Å. In this paper, we provide
structural and mechanistic models for head-to-head terpene
synthase activity, and inhibition, based on nine X-ray structures;
studies of site-directed mutagenesis based on both structure and
bioinformatics; chemical reactivity; and comparisons with head-
to-head terpene synthases and terpene cyclases.
(
PSPP), the dehydrosqualene (DHS) product, as well as a series of
inhibitors. The results indicate that, on initial diphosphate loss, the
primary carbocation so formed bends down into the interior of the
protein to react with C2,3 double bond in the prenyl acceptor to
form PSPP, with the lower two-thirds of both PSPP chains occupy-
ing essentially the same positions as found in the two farnesyl
chains in the substrates. The second-half reaction is then initiated
by the PSPP diphosphate returning back to the Mg cluster for
ionization, with the resultant DHS so formed being trapped in a
surface pocket. This mechanism is supported by the observation
that cationic inhibitors (of interest as antiinfectives) bind with
their positive charge located in the same region as the cyclopropyl
carbinyl group; that S-thiolo-diphosphates only inhibit when in
the allylic site; activity results on 11 mutants show that both
DXXXD conserved domains are essential for PSPP ionization; and
the observation that head-to-tail isoprenoid synthases as well as
terpene cyclases have ionization and alkene-donor sites which
spatially overlap those found in CrtM.
2þ
triterpene ∣ X-ray crystallography ∣ drug discovery ∣ staphyloxanthin ∣
quinuclidine
Results and Discussion
Structure of PSPP Bound to CrtM. The overall reactions catalyzed by
CrtM, SQS, and PSYare shown in Fig. 1 and involve ionization of
one isoprenoid diphosphate molecule to form a 1′ carbocation,
which then undergoes nucleophilic attack by the C2,3 double bond
in the second isoprenoid diphosphate to form, after proton loss,
PSPP or PPPP. After a second ionization, these molecules then
undergo a complex series of rearrangements (12) thought to in-
volve cyclopropyl and cyclobutyl species to form either primarily
dehydrosqualene (with CrtM; or SQS in the absence of NADPH);
squalene (with SQS, in the presence of NADPH), or phytoene
(with PSY). But which site houses the cation (donor), and which
ead-to-head terpene synthases catalyze the first committed
H
steps in the biosynthesis of sterols and carotenoid pigments:
the C1′-2,3 condensation of two isoprenoid diphosphates to form
a cyclopropylcarbinyl diphosphate (1, 2), followed by ring open-
ing to form squalene, dehydrosqualene, or phytoene. In humans
and in many pathogenic yeasts, fungi, and protozoa, as well as in
plants, the isoprenoid diphosphate is farnesyl diphosphate (FPP)
and the initial product is the C30 isoprenoid, presqualene diphos-
phate (PSPP). As implied by its name, PSPP is then converted (by
the same enzyme as used in the condensation reaction, squalene
synthase, SQS) to squalene which, after epoxidation, is cyclized
to lanosterol (3), as shown in Fig. 1. Lanosterol then undergoes
numerous additional reactions, resulting in formation of sterols,
key cell membrane components. As such, squalene synthase
inhibitors are of interest as antiparasitics, in particular against
Trypanosoma cruzi (4) and Leishmania spp. (5), the causative
agents of Chagas disease and the leishmaniases. In plants, the
related enzyme phytoene synthase (PSY) catalyzes the condensa-
tion of two C20 isoprenoid diphosphate (geranylgeranyl diphos-
phate, GGPP) molecules (6) to form prephytoene diphosphate
Author contributions: F.-Y.L., C.-I.L., Y.-L.L., R.C., A.H.-J.W., and E.O. designed research;
F.-Y.L., C.-I.L., Y.-L.L., Y.Z., K.W., W.-Y.J., T.-P.K., R.C., and E.O. performed research; F.-Y.L.,
C.-I.L., Y.-L.L., W.-Y.J., T.-P.K., R.C., and E.O. analyzed data; Y.Z. and K.W. contributed
new reagents/analytic tools; and A.H.-J.W. and E.O. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Research Collaboratory for Structural Bioinformatics Protein Data Bank, www.pdb.org for
dehydrosqualene synthase complexed with presqualene diphosphate (PDB ID codes 3LGZ,
3
ADZ, 3NPR), dehydrosqualene (PDB ID code 3NRI), BPH-651 (PDB ID code 3ACW), BPH-673
(
PDB ID code 3ACX), BPH-702 (PDB ID code 3ACY), and GGSPP (PDB ID code 3AE0), and for
(
PPPP) that, after ring opening, forms phytoene, which is then
human squalene synthase complexed with BPH-652 (PDB ID code 3LEE).
converted to carotenoid pigments (7) (Fig. 1). In the bacterium
Staphylococcus aureus, the initial step in formation of the caro-
tenoid pigment staphyloxanthin (STX) is carried out by conden-
sation of two FPP molecules by the enzyme dehydrosqualene
synthase (CrtM), again initially forming PSPP (8), which is then
1
F-Y.L. and C-I.L. contributed equally to this work.
2
To whom correspondence may be addressed. E-mail: eo@chad.scs.uiuc.edu or ahjwang@
gate.sinica.edu.tw.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1010907107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1010907107
PNAS ∣ December 14, 2010 ∣ vol. 107 ∣ no. 50 ∣ 21337–21342

Fig. 1. Schematic illustration of the reactions catalyzed by head-to-head terpene synthases: CrtM, SQS, and PSY. All reactions involve an initial C1′-2,3 cyclo-
propanation step. The end products of the biosynthetic pathways are highly varied and include sterols (cholesterol, ergosterol) and carotenoids (staphylox-
anthin, β-carotene).
is the alkene (acceptor)? How do PSPP and dehydrosqualene
DHS) bind to CrtM (or SQS)? And where do the diphosphates
protein, which is known to be unreactive (13). The Y129A CrtM
mutant was indeed unreactive, with either FPP (0% activity) or
PSPP (7% activity, Fig. S1) as substrate, indicating that both the
first-half (PSPP formation) as well as the second-half (PSPP to
dehydrosqualene) reactions were inhibited, which enabled us to
obtain diffraction quality crystals of a CrtM/PSPP complex. Our
initial CrtM/PSPP crystals diffracted to 2.4 Å (full crystallographic
data and structure refinement details are given in Table S1), and
using the CrtM-FSPP structure (PDB ID code 2ZCP) minus
ligands as a template, we obtained the electron density results
shown in Fig. S2A (PDB ID code 3LGZ). We then obtained a sec-
ond structure (electron density shown in Fig. 2B) with improved
(
ionize? In our work on CrtM (10), we used the nonreactive
substrate analog, S-thiolo-farnesyl diphosphate (FSPP), to define
two prenyl diphosphate binding sites: S1 and S2. The structure
obtained (PDB ID code 2ZCP) is shown in Fig. 2A from which
it can be seen that the C1′-C2,3 distances for the two possible
mechanistic models (i.e., S1 ¼ donor or S1 ¼ acceptor) are very
similar (5–5.4 Å) making it impossible to make reliable donor–
acceptor site assignments based solely on this metric.
We therefore produced a CrtM mutant (Y129A) that, based on
sequence alignment with rat SQS, corresponds to Y171 in the rat
Fig. 2. Crystallographic results for CrtM with a bound substrate-like inhibitor FSPP and the intermediate PSPP. (A) Bound FSPP (PDB code ID 2ZCP), from ref. 10.
B) The 2Fo-Fc map for PSPP contoured at 1σ (green) and 3σ (red) (PDB code ID 3ADZ). (C) Superposition of PSPP (PDB ID code 3ADZ) on FSPP in CrtM. (D) Close-
up view of PSPP-FSPP superposition. Arrows indicate movements of the C1′, C2, and C3 atoms.
(
2
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Lin et al.

2
þ
(
1.89 Å) resolution at a higher [Mg ] (PDB ID code 3ADZ; full
crystallographic data and structure refinement details are given in
Table S1; crystallization details are in SI Methods), Fig. 2C. Both
α
structures (which have a 0.3-Å protein C and 0.7-Å ligand rmsd)
are shown superimposed in Fig. S2C. In addition, we obtained
a third PSPP structure using wild-type CrtM [crystallized in the
presence of 0.2 M sodium tartrate (SI Fig. S2 B and D, and
Table S1)], which had a 0.18, 0.45-Å protein and 0.19, 0.8-Å ligand
rmsd versus the two mutant structures, indicating all three struc-
tures are virtually identical.
What is immediately obvious from these structures, shown
in cyan) superimposed on the CrtM/FSPP structure (in green
(
and yellow) in Fig. 2C, is that (i) the diphosphate (PPi) group
seen in the S1 FSPP site is no longer present, indicating that the
FPP in this site is the prenyl donor (which ionizes to form the
1
′-carbocation); (ii) the S1 FPP C1′ moves down by ∼2.5 Å to
form C1′ in PSPP, while (iii) the S2 FPP C2 and C3 move up by
1.5 Å, to form C2,3 in the cyclopropane ring of the PSPP pro-
∼
duct; (iv) the S2 PPi group moves “up” somewhat (on average, the
non-H atoms move by ∼1.8 Å), but overall this group remains
very close to its original position; and (v) the lower two-thirds
of the PSPP side chains hardly move at all with respect to their
positions in FSPP, only a ∼1.2-Å rmsd for 27 carbon atoms (0.7 Å
for 25 carbons). Also of interest is the observation that the PSPP
side chain in S1 is highly “bent” and, although it appears shorter,
is actually the longer one (11 versus 9 contiguous carbons),
whereas the S2 chain is quite straight, occupying the same site
as the biphenyl ring-containing inhibitors reported previously
(
10). These results strongly support a “first-half” reaction me-
chanism in which FPP in S1 ionizes to form the primary carboca-
tion which then moves down to react with the C2,3 double bond
Fig. 3. Effects of FSPP on CrtM catalysis and a GGSPP inhibitor structure. (A)
Rate of PPi generation increases with FSPP concentration (error bars are from
duplicate data points, R2 ¼ 0.85). (B) Rate of dehydrosqualene formation de-
þ
in the FPP in S2 to form (after H abstraction), PSPP, with the
highly conserved Asp residues in the first DXXXD domain being
essential for catalysis (Fig. S1). To further test this mechanistic
proposal, we next investigated the structure and activity of
a series of S-thiolo-diphosphates interacting with CrtM.
2
creases with FSPP concentration (duplicate, R ¼ 0.78). These results indicate
“
dead-end” formation of S-thiolo-PSPP, as confirmed by mass spectrometry.
(
(
C) The 2Fo-Fc map (contoured at 0.8σ and 3σ) of GGSPP bound to F26A CrtM.
D) GGSPP (green and yellow) binds to both S1, S2; would clash with F26 (in
red) if present; FSPPs are in cyan.
Structure and Activity of Substrate Analogs. In our previous work
on CrtM, we used S-thiolo-FSPP as an unreactive substrate
analog (10). The lack of activity of other S-thiolo-isoprenoid
diphosphates as allylic/cationic prenyl donors has been studied
extensively by Phan et al. (14) who found that, e.g., S-thiolo-
dimethylallyl diphosphate (DMAPP) inhibits farnesyl dipho-
sphate synthase (FPPS) (because it does not ionize, in the allylic
site, S1), whereas S-thiolo-isopentenyl-diphosphate (IPP) inhibits
FPPS, because it forms S-thiolo-geranyl diphosphate (GPP),
which again cannot ionize, in S1 (14). Plus, Hosfield et al. (15)
have shown that S-thiolo-DMAPP binds to the allylic (S1) site
in FPPS, as does S-thiolo-FPP in geranylgeranyl diphosphate
synthase (GGPPS) (16). These findings lead to the idea that other
S-thiolo-isoprenoid diphosphates might be interesting probes of
CrtM structure and function.
and S-thiolo diphosphates [representative liquid chromatography
(
LC)-MS results are shown in Figs. S4 and S5 and are summar-
ized in Table S2]. The results can be summarized as follows: Only
FPP (in the S1 site) can ionize, whereas FPP, GGPP, FSPP, or
S-thiolo-geranylgeranyl diphosphate (GGSPP) (in S2) can all
provide the alkene, prenyl acceptor, in WT CrtM. So, longer
chains (C20) can fit in S2, but not in S1, because we find no
activity (PPi release) with GGPP as substrate. However, in an
F26A mutant, GGPP actually reacts to form phytoene (18), be-
cause the bulky F26 (at the “bottom” of the S1 binding site)
that blocks GGPP binding to S1 is removed, and the reaction
can proceed. If this idea is correct, it should be possible to bind
two molecules of GGSPP to the F26A CrtM mutant. This is
indeed the case, as shown in Fig. 3 C and D where we see that
the S1 side chain in the CrtM/GGSPP complex (PDB ID code
3AE0; Table S1) would clash with F26, shown in red (if it were
present). These results are all consistent with S1 being the ioniza-
tion site for FPP in PSPP formation.
In order to see how FSPP might inhibit the conversion of
FPP to PSPP, and then dehydrosqualene, we carried out a series
of CrtM inhibition experiments. Remarkably, we found that the
rate of the CrtM reaction (monitored in this case via PPi release)
increased with FSPP concentration (Fig. 3A). When we assayed
for DHS production, we found that its formation was being
inhibited (Fig. 3B), so it seemed that FSPP must be reacting
with FPP to form a nonreactive S-thiolo-PSPP, in much the same
way that S-thiolo-IPP can react with DMAPP in FPPS to form
S-thiolo-GPP (14). To verify formation of S-thiolo-PSPP, we
isolated the product of the reaction chromatographically, then
carried out a reductive cleavage (17) and obtained the mass spec-
trometry results shown in Fig. S3, which confirm formation of
the C H S product, presqualene thiol.
A Catalytic Model for Dehydrosqualene Synthase. These results
all support the initial reaction model shown in Fig. 4 (and
Fig. S6) in which the S1 FPP ionizes to form the 1′-carbocation,
Fig. 4 A and B. This carbocation then moves down into the inter-
ior of the protein to react with C2,3 in the S2 FPP, Figs. 2D
and 4B, with remarkably little change in the conformation of the
lower two-thirds of the FSPP and PSPP hydrocarbon chains
(Fig. 2C). The second PPi in PSPP then moves up (Fig. 4C) into
3
0
50
2þ
To investigate this topic in more detail, we next investigated
the reactions between farnesyl and geranylgeranyl diphosphates
the S1 site to interact with the three Mg , and the cyclopropyl-
carbinyl cation so formed (Fig. 4 D and E) then rearranges,
Lin et al.
PNAS
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Fig. 4. Schematic illustration of the conversion of FPP to dehydrosqualene catalyzed by CrtM, based on the crystallographic results. (A) Bound FPP in S1 ionizes.
B) Carbocation reacts with S2 FPP alkene group. (C) PSPP PPi in S2 moves to S1 site. (D) PSPP PPi ionizes. (E and F) Carbocation rearranges and after proton loss
(
forms final product, dehydrosqualene. (G and H) Two possible condensation reactions, in S1, S2. Only that shown in G is consistent with the crystallographic
results.
as proposed (12), forming DHS (Fig. 4F), which we find binds to
the surface pocket shown in Fig. 5 A (PDB ID code 3NRI) (full
crystallographic data and structure refinement details are given in
Table S1; crystallization details are in SI Methods). Chemical
structures are shown for clarity in Fig. 4G. The alternate model
second-half reactions. This observation strongly supports the
ionization of both FPP as well as PSPP in the same site—the
2
þ
3
Mg
trimetal cluster observed in the FSPP structure (Fig. 2A).
Based on the observation that Y129 is essential for both half-
reactions, that there are no conformational changes between
WT and the Y129A mutant, together with the observation that
Y129 is ∼8 Å from the closest FPP, it seems that the most likely
role for this highly conserved residue is to facilitate removal of the
(Fig. 4H), in which the S2 site FPP ionizes initially, results in
the longer (11 contiguous carbons) PSPP side chain occupying
the S2 product site, which is not seen experimentally.
But which residues are involved in the second-half reaction?
One possibility is that the PSPP diphosphate just ionizes in
2þ
Mg ∕PPi product complex formed in both half-reactions from
the enzyme.
2
þ
the S2 site and there is indeed one Mg that interacts with the
PSPP PPi group. The second possibility is that the PSPP dipho-
sphate group could simply flip back up to the two DXXXD∕
Structural Basis for CrtM/SQS Inhibition by Cationic Species. Next, we
consider CrtM and SQS inhibition. One of the most potent classes
of SQS inhibitor are the quinuclidines (or quinuclidinols), com-
pounds such as BPH-651, and E5700 and ER119884 (from Eisai
Pharmaceuticals) (Scheme 1), which have been found to have low
nanomolar activity against both T. cruzi and human SQS (4, 5, 22),
as well as potent activity against T. cruzi cell growth (5). These
compounds are of interest as antiinfectives, but nothing is known
structurally about how they might act. The 3-OH quinuclidine pKa
is expected to be ∼10–11 (23), so these compounds are likely to
bind to SQS as cationic (ammonium) species, and it seemed pos-
sible that, as with, e.g., nitrogen-containing bisphosphonate inhi-
bitors of FPPS (24, 25), they might mimic the transition state–
reactive intermediates involved in PSPP (or squalene–dehydros-
qualene) formation. Although BPH-651 is only a modest CrtM
2þ
Mg
domain (Fig. 5B) to undergo this second ionization, as
3
illustrated in the Glide (19) docking pose shown in Fig. 5C. To
help distinguish between these two possibilities, we used the
JPRED3 (20) and SCORECONS (21) programs to rank order
the essential residues in CrtM (excluding structural Gly, Ala, and
Pro), then mutated nine of these to Ala and tested their activity in
both first-half (FPP to PSPP) and second-half (PSPP to DHS)
reactions. Results are shown in Table S1. What is clear from these
results is that Y129 as well as all of the top ranked Asp residues
(
D48 and D52 in the first Asp-rich region; D172 and D176 in the
second Asp-rich domain; Fig. 5B) are essential for both first- and
inhibitor (K ¼ 17.5 μM), we were able to obtain the structure
i
of a CrtM/BPH-651 complex (full crystallographic details are
given in Table S3), as well as that of a second ammonium-contain-
Fig. 5. Dehydrosqualene structure, important residues in catalysis, and PSPP
ionization model. (A) The 2Fo-Fc map for dehydroqualene (1σ, magenta)
bound to CrtM surface pocket, superimposed on PSPP (in cyan). (B) Key cat-
alytic residues, D48, D52, and R45 are essential for second-half reaction and
indicate PPi ionization in S1. (C) Proposed movement of PSPP PPi into S1 for
ionization; PSPP is in gray and the proposed ionization conformation of PSPP
is in cyan. The ionization conformation is obtained by using Glide program
(
19), docking to PDB ID code 2ZCP.
Scheme 1.
2
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Lin et al.

ing species (26) BPH-673 (Table S3), a potent SQS inhibitor which
substituent that makes them particularly potent. So how might
their bulky side chains bind? To help answer that question, we
first need to determine how good a model for SQS is CrtM
(and vice versa).
has a K ∼ 2 μM for CrtM inhibition. Both compounds contain
i
biphenyl side chains and bind into the “linear” S2 site, as shown
in Fig. 6 A and B. In each case, the positively charged center (blue)
is located quite near the cyclopropyl ring in PSPP (BPH-651, 1-Å
closest distance; BPH-673, 2 Å), with the superpositions shown in
Fig. 6 A and B clearly suggesting that both cationic inhibitors act as
transition state–reactive intermediate analogs which occupy the
S2 site, mimicking a cyclopropyl carbocation species, rather than
being isosteres for the farnesyl carbocation which forms in S1. In
FPPS, the cationic or nitrogen-containing bisphosphonate class of
inhibitors bind to the S1 site (18, 21, 22), but of course most of the
binding energy there can be attributed to the bisphosphonate–
We therefore next obtained the X-ray crystallographic struc-
ture of BPH-652, the CrtM inhibitor whose structure we reported
previously (10), bound this time to human SQS (PDB ID code
3
LEE; full crystallographic details are given in Table S3). Using
the CrtM/SQS protein structure alignment (Fig. 6C), we find
that there is a 2.0-Å rmsd between the BPH-652 ligand heavy
atoms coordinates, showing close structural similarity. The rmsd
reduces to 1.2 Å when the alignment is based solely on the con-
served Asp residues, or 0.6 Å when the alignment is based solely
on the ligands. Then we obtained the X-ray structure of an analog
of BPH-652, BPH-702 (PDB ID code 3ACY), which contains the
benzyl substituent found in the most potent quinuclidinol inhibi-
tors. As can be seen in Fig. 6D, the phosphonosulfonate BPH-702
backbone binds in the S1 PPi-binding domain (seen in the FSPP
structure); the biphenyl ether side chain binds in the S2 side-chain
region, whereas the benzene ring in the benzyl group is located at
the terminus of the S1 side-chain in the FSPP structure (Fig. 6D).
When we superimpose the quinuclidinol (BPH-651) and benzyl
biphenyl phosphonosulfonate (BPH-702) structures (Fig. 6E), we
see that the ER119884 side chain can readily fit into the space
occupied by these two compounds, and a docking pose (using
Glide) (19) for this potent inhibitor (docked to the CrtM/PSPP
protein structure) is shown in Fig. 6F. Also of interest is the
observation that the cationic center in the quinuclidinol overlays
the PSPP cyclopropane ring (Fig. 6F), suggesting that quinuclidi-
nol may act as an isostere for a cyclopropyl carbinol carbocation
reactive intermediate.
2
þ
3
Mg
interaction. In the cationic inhibitors here, no such metal
ion chelation is present, and binding is dominated by hydrophobic
interactions with, potentially, the ammonium groups (and the
cationic reaction intermediates) also being stabilized by the pro-
tein’s charge field, just as in FPPS (17). The Eisai SQS inhibitors
(E5700, ER119884) do, however, both have an additional benzyl
Comparisons with Head-to-Tail Terpene Synthases and Terpene
Cyclases. These results lead to the following question: Do the
head-to-head, head-to tail, and terpene cyclases all have the same
basic “S1, S2” structure and function? To help answer this ques-
tion, we compared the CrtM/FSPP structure (PDB ID code
2
ZCP) with the 3D structures of a series of other prenyl trans-
ferases: FPPS (27), GGPPS (16), epiaristolochene synthase (28),
and limonene synthase (29), where the donor–acceptor site func-
tions are well known to deduce, based on structural homology,
which FPP in CrtM loses diphosphate (to form the cationic, pre-
nyl donor), and which acts as the nucleophile or prenyl acceptor.
We first aligned the head-to-tail prenyl synthase FPPS, containing
zoledronate and IPP (PDB ID code 2F8Z) to the CrtM/FSPP
structure using the combinatorial extension program (30). The
superposition (Fig. S7A) indicates that the FSPP diphosphate in
S1 (green in Fig. S7A) is close to the bisphosphonate group in the
allylic (cationic) site in FPPS, whereas the IPP diphosphate (and
the IPP double bond) is in S2. The same conclusion is reached
with FSPP in GGPPS (Fig. S7B), the IPP molecule again being
located in the S2 or prenyl acceptor site (yellow in Fig. S7B). So,
these results indicate that the S2 FPP acts as the nucleophile in
CrtM (and by analogy, in SQS and PSY). Similar results are
found on comparison of the CrtM structure with those of the class
I (ionization initiated) terpene cyclases epiaristolochene synthase
(Fig. S7C) and limonene synthase (Fig. S7D) with bound inhibi-
tors. In both cases, the phosphonate or diphosphate headgroups
Fig. 6. X-ray crystallographic structures of inhibitors bound to CrtM.
A) BPH-651, a 3-OH quinuclidine (PDB ID code 3ACW). (B) BPH-673 (PDB ID
(
code 3ACX), an biphenyl/ammonium inhibitor. Both structures are shown
superimposed on PSPP (PDB ID code 3ADZ, in cyan). (C) Comparison between
BPH-652 bound to CrtM (orange; PDB ID code 2ZCQ) and SQS (green; PDB ID
code 3LEE). The heavy atom rmsd in 2.1 Å but this distance reduces to 1.2 Å if
the structures are aligned using the conserved Asp residues (or 0.6 Å for the
ligands alone). The apparent “offset” may be related to an NADPH binding
site in SQS. (D) BPH-702 (orange) bound to CrtM superimposed on the FSPP
structure (in green, yellow). Note that the phosphonosulfonate group binds
in S1, the biphenyl ether side chain in S2, while the benzyl substituent also
binds in S1. (E) BPH-702 (the benzyl inhibitor, in orange) superimposed on
the quinuclidine BPH-651. (F) Docking pose for the quinuclidine ER119884
2
þ
bind in or close to the S1 diphosphate-Mg cluster seen in CrtM,
whereas the side chains are in, or close to, the S2 site. Because it is
clear that S1 in the cyclases has to be involved in ionization while
the side chain in S2 site acts as the nucleophile, we again deduce
that the S1 site in CrtM is the cationic (prenyl donor) site,
whereas S2 is the (olefinic) prenyl acceptor site.
Conclusions
Overall, the results presented above are of broad general interest
because they provide detailed insights into the first committed
(
in yellow) bound to CrtM, superimposed on PSPP (in cyan).
Lin et al.
PNAS
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December 14, 2010
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vol. 107
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reactions in sterol and carotenoid biosynthesis: the head-to-head
condensation of two prenyl diphosphates to form a cyclopropyl-
carbinyl diphosphate, followed by ring opening to form DHS.
We conclude that FPP in the bent S1 site ionizes to form the
prenyl donor carbocation which then moves into the protein’s
interior to react with the S2 site FPP C2,3 double bond, forming
PSPP, completing the first-half reaction. In the second-half reac-
tion, the PSPP diphosphate then moves back to S1 to ionize. Two
cationic inhibitors act as PSPP–carbocation isosteres, binding
with their charge center in the same region as the cyclopropyl
group, whereas their side-chain substituents bind also to the S1
side-chain site. The same S1 þ S2 binding motif is seen with a
potent phosphonosulfonate, and dual-site binding is likely to be
the origin of the potent activity of the quinuclidinols. This infor-
mation should be of interest in the context of developing CrtM/
SQS inhibitors as antiinfective drugs.
PSPP was prepared using a similar procedure, except that FSPP and FPP
2∶1) were used. LC/MS analyses were carried out using an Agilent LC/MSD
Trap XCT Plus instrument. Compounds were separated on a 5-μm (4.6×
(
1
50 mm) Eclipse XDB-C8 column (Agilent) using a 0–100% acetonitrile (in
2
5 mM NH4HCO3 buffer) gradient and monitored by using negative-ion
mode electrospray ionization.
CrtM crystals were obtained as described previously (10) with some
modifications and were soaked with PSPP and various inhibitors to obtain
the complex structures. Full details are given in SI Methods. Structure deter-
minations and refinements were carried out as described previously (10) with
full details given in SI Methods.
ACKNOWLEDGMENTS. We thank Dr. Howard Robinson for collecting the
hSQS X-ray data, M.-F. Hsu for technical assistance, Mr. Y.G. Gao for help with
crystallization, Dr. Dolores Gonzalez-Pacanowska for providing the hSQS
expression system, and R. M. Coates for a sample of DHS. This work was
supported by grants from the United States Public Health Service [National
Institutes of Health Grant AI-074233 (to E.O.)] and from the Academia Sinica
and the National Science Council [NSC 97-3112-B-001-017 and NSC 98-3112-
B-001-024 (to A.H.-J.W.)]. Portions of the research were carried out at
the National Synchrotron Radiation Research Center, a national user facility
supported by the NSC of Taiwan, Republic of China, and the Photon Factory
in Japan. Use of the Advanced Photon Source was supported by the US
Department of Energy, Office of Science, Office of Basic Energy Sciences,
under Contract DE-AC02-06CH11357. Use of the Life Science Collaborative
Access Team Sector 21 was supported by the Michigan Economic Develop-
ment Corporation and the Michigan Technology Tri-Corridor for the support
of this research program (Grant 085P1000817). Data for the hSQS study were
measured at beamline X29A of the National Synchrotron Light Source, where
financial support comes principally from the Offices of Biological and Envir-
onmental Research and of Basic Energy Sciences of the US Department of
Energy, and from the National Center for Research Resources of the National
Institutes of Health.
Methods
FPP, FSPP, GGPP, and GGSPP were synthesized according to literature methods
(
14, 31). PSPP was made biosynthetically by using human SQS (hSQS). Fifty
milligrams of hSQS, 10 mg of FPP, together with 20 units of baker’s yeast
pyrophosphatase (Sigma-Aldrich) were dissolved in 10 mL reaction buffer
(
25 mM Hepes, 100 mM NaCl, 0.5 mM MgCl , pH7.4). The reaction mixture
2
was stirred at room temperature for 8 h, then quenched by adding 10 mM
EDTA and solid NaCl. The solution was centrifuged at 4;000 × g for 20 min and
the supernatant was applied to a 5-mL C8 solid-phase extraction column
(
1
SC300C8K, Western Analytical Products, Inc.). The column was washed with
0 mL reaction buffer and 10 mL 30% acetonitrile, sequentially, to remove
residual enzyme, substrate, and other contaminants. PSPP was eluted using
0% acetonitrile (in water) and purity was confirmed by LC-MS/MS. S-thiolo-
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Lin et al.