Thiol-modifying inhibitors for understanding squalene cyclase function
Paola Milla
1
, Alexander Lenhart
2
, Giorgio Grosa
3
, Franca Viola
1
, Wilhelm A. Weihofen
2
, Georg E. Schulz
2
and Gianni Balliano
1
1
Universita
`
degli Studi di Torino, Dipartimento di Scienza e Tecnologia del Farmaco, Torino, Italy;
2
Universita
¨
t Freiburg,
Institut fu
¨
r Organische Chemie und Biochemie, Freiburg, Germany;
3
Universita
`
degli Studi del Piemonte Orientale ÔA. Avogadro,
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Novara, Italy

squalene-maleimide proved to be a very effective time-
dependent inhibitor.
Keywords: Alicyclobacillus acidocaldarius;membrane
protein; site-directed mutagenesis; squalene cyclase; thiol
reagents.
Oxidosqualene cyclases (OSCs) and squalene-hopene
cyclases (SHCs) are key enzymes in triterpenoid biosynthesis:
they transform acyclic isoprenoid precursors into tetra- and
pentacyclic compounds [1]. OSCs can be considered
taxonomic markers, as they catalyse the conversion of 2,3-
oxidosqualene into lanosterol in nonphotosynthetic organ-
isms (fungi and mammals), and into cycloartenol and other
tetra- and pentacyclic triterpenes in plants [2]. In prokary-
otes, SHCs convert squalene into hopene or diplopterol
(Fig. 1), pentacyclic triterpene precursors of hopanoids.
These compounds are thought to have functions similar to
those of sterols in eukaryotic membranes [3].
An important contribution to the understanding of the
catalytic mechanisms controlled by OSCs and SHCs came
from the crystal structure of SHC from Alicyclobacillus
acidocaldarious [4,5]. X-ray analysis revealed a membrane
protein with membrane-binding characteristics similar to
those of two prostaglandin-H
2
synthase isoenzymes [6,7].
These membrane proteins are called monotopic as they are
shaped so as to submerge from one side of the membrane
into the nonpolar part of the phospholipid bilayer without
protruding through it [8]. The enzyme has a hydrophobic
plateau plunging into the lipophilic centre of the membrane.

,sodium
borotritiure (Ph)
3
P, triphenylphosphine,
Enzymes: oxidosqualene cyclase (EC 5.4.99.7); squalene-hopene
cyclase (EC 5.4.99.x).
Note: P. Milla and A. Lenhart contributed equally to this work.
Note: a web site is available at
/>(Received 1 November 2001, revised 18 February 2002, accepted 25
February 2002)
Eur. J. Biochem. 269, 2108–2116 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02861.x
critically located Cys residues, either present in native
protein or inserted by site-directed mutagenesis, were
labelled with different thiol-reacting molecules, designed
and synthesized in our laboratories.
MATERIALS AND METHODS
NMR and MS of chemical products
1
H-NMR spectra were recorded on a Jeol EX-400 or Jeol
GX-270, with SiMe
4
as internal standard. Mass spectra
were obtained on a VG Analytical 7070 EQ-HF spectro-
meter by electron impact ionization. IR and UV spectra
were recorded, respectively, on Perkin-Elmer 781 and
Beckman DU 70 spectrophotometers.
Chemicals
Light petroleum refers to the fractions of bp 40–60 °C.
Tetrahydrofuran was distilled under sodium benzophenone
ketyl. Silica gel was 70–230 mesh. Squalene was from

for 3 h on ice while a precipitate appeared. CHCl
3
(60 mL)
was then added and the solution was washed with 5%
NaHCO
3
(2 · 120 mL) and saturated brine (40 ng NaCl in
100 mL H
2
O; 1 · 100 mL). The organic phase was dried
over anhydrous sodium sulfate and evaporated in vacuo.
The crude product was purified by flash-chromatography
using CHCl
3
as eluant to give 1.36 g CPTO (63% yield).
ESI-MS m/z:213(M
+
, 11), 195 (100), 185 (8), 137 (43);
1
H-
NMR (CDCl
3
) d:3.14(m,1H,5-H
a
), 3.36 (m, 1H, 5-H
b
),
4.68 (m, 1H, 4-H
a
), 4.91 (m, 1H, 4-H

sulfate the solvent was evaporated under reduced pressure.
The crude product was purified by column chromatography
on silica with chloroform as eluant to give 1.05 g N,N¢-
(dithiodi-2,1-ethanediyl)bis 4-chlorobenzamide (68% yield).
ESI-MS m/z: 428 (M
+
, < 1), 215 (33), 182 (60), 139 (100),
111 (39);
1
H-NMR (CDCl
3
/CD
3
OD) d:2.81(t,4H,-CH
2
-
S), 3.57 (t, 4H, -CH
2
-N), 7.23 (d, 4H, aromatic protons),
7.61 (d, 4H, aromatic protons); IR (KBr) m
max
: 3302, 3236,
1638, 1628, 1597, 1541, 1489Æcm
)1
;UV(CH
3
OH) k
max
: 204,
235.

GRASP
[9].
Ó FEBS 2002 Effects of SH-modifying agents on squalene cyclase (Eur. J. Biochem. 269) 2109
sodium sulfate the solvent was evaporated under reduced
pressure. The crude product was purified by column
chromatography on silica with CHCl
3
andthenCHCl
3
/
CH
3
OH 98 : 2 to give DTS (5) (220 mg, 53% yield). ESI-
MS m/z: 429 (M ± 16, 54), 214 (29), 182 (44), 156 (35), 139
(100), 111 (54);
1
H-NMR (CD
3
OD) d:3.57(m,4H,-CH
2
-
SO and -CH
2
-S), 3.82–3.93 (m, 4H, -CH
2
-N), 7.53 (m, 4H,
aromatic protons), 7.88 (m, 4H, aromatic protons); IR
(KBr) m
max
: 3680, 3412, 2920, 1664, 1597, 1541, 1480Æcm

above the origin with n-hexane/ethyl acetate (90 : 10; v/v).
Radioactive areas corresponding to squalene and 2,3-oxido-
squalene were scraped off and eluted with dichloromethane.
The solvent was dried under N
2
and [
14
C]-squalene and
[
14
C]-3S-2,3-oxidosqualene were dissolved in benzene. The
radiochemical purity of products was evaluated by scanning
TLC plates with a System 2000 Imaging Scanner (Packard).
Radioactivity was measured by Liquid Scintillation Count-
ing (Beckman).
All of the radiolabelled compounds were compared
chromatographically with authentic radio-inert samples.
Determination of the radioactive substances and isotope
counting were carried out as already described [15,16].
Radiolabelled CNDT-squalene (1) was synthesized via
the following steps (Fig. 3): (i) synthesis of [1-
3
H]trisnor-
squalene alcohol; (ii) synthesis of [1-
3
H]trisnorsqualene
thioacetate; (iii) synthesis of [1-
3
H]trisnorsqualene thiol;
(iv) transformation of [1-

H]trisnorsqualene alcohol. The product was
purified, after dissolution with light petroleum, by column
chromatography on silica gel with 100% light petroleum to
remove impurities, then light petroleum/diethylether 90 : 10
to give 18 mg (0.046 mmol) of pure [1-
3
H]trisnorsqualene
alcohol. The radiochemical purity of the alcohol was
determined by radiochromatogram with light petroleum/
diethylether 80 : 20 and then revealed with iodine vapour.
Total activity: 4.2 mCi; specific activity: 92 mCiÆmmol
)1
;
chemical yield: 88%.
(ii) [1-
3
H]Trisnorsqualene thioacetate: [1-
3
H]-(4E,8E,
12E,16E) S-[4,8,13,17,21-pentamethyl-4,8,12,16,20-docos-
apentaenyl] thioacetate. A solution of diisopropyl azodi-
carboxylate (71.6 mg, 0.354 mmol) in 0.5 mL anhydrous
tetrahydrofuran was added to a well-stirred solution of
triphenylphosphine (92.5 mg, 0.350 mmol) in 3 mL anhy-
drous tetrahydrofuran at 0 °C. The mixture was stirred at
0 °C for 30 min and produced a white precipitate. A solu-
tion of [1-
3
H] trisnorsqualene alcohol (18 mg, 0.046 mmol)
and thiolacetic acid (37.2 mg, 0.488 mmol) in 0.5 mL

under nitrogen. The mixture was stirred 25 min at room
temperature then 25 min under reflux. LiAlH
4
excess was
destroyed by the careful addition of 7 mL 1
M
HCl solution.
The ether layer was separated and the aqueous phase
extracted with dichloromethane. The combined organic
phases were dried over sodium sulfate and the solvent
evaporated under reduced pressure. The crude product was
used without purification for the following step.
(iv) [1-
3
H]Trisnorsqualene nitrobenzoic acid (1): [1-
3
H]-
6-nitro-3-[(4E,8E,12E,16E)-4,8,13,17,21-pentamethyl-4,8,12,
16,20-docosapentaenyldisulfamyl] benzoic acid; 5,5¢-dithio-
bis(2-nitrobenzoic acid) (41 mg, 0.103 mmol) was dissolved
Fig. 3. Scheme for the synthesis of [1-
3
H] CNDT-squalene. The asterisk
indicates position of
3
H-label: (i) NaB
3
H
4
(ii) (Ph)

¨
tTu
¨
bingen) [18].
For production of recombinant SHC, the overexpression
system described elsewhere [19] was used. Mutants D376C/
C435S, C455S and C50S had been generated for structure
analysis [5] utilizing the phosphorothioate method (Sculp-
tor
TM
, Amersham).
To generate the quadruple mutant, gene fragments
containing mutations C455S and C50S were introduced
into expression plasmid pKSHC1 using restriction sites
SacI/HindIII and EcoRI/ApaI, respectively. Mutations
C25S and C537S were created with the megaprimer method
[20,21]. The first round of PCR amplification was per-
formed with Pwo polymerase (Peqlab, Erlangen, Germany)
and primers MP-C25S and MP-ApaI or MP-C537S and
MP-HindIII, respectively. The PCR products were purified
by agarose gel electrophoresis and gel extraction (QIAquick
`
Gelextraction-Kit, Qiagen) and were used as ÔmegaprimersÕ
in the second round of PCR with the additional primers
MP-EcoRI or MP-SacI. Sequences of the synthetic
oligonucleotides (MWG, Ebersberg, Germany) were as
follows (with changes from the wild-type sequence under-
lined): Mp-C25S, 5¢-CTCCTCTCC
AGCCAAAAGG-3¢;
MP-C537S, 5¢-GGCGAGGAC

nonsaponifiable lipids were extracted twice with 1 mL
petroleum ether. The extract was chromatographed on silica
gel plates developed in petroleum ether. The radioactivities
of squalene, hopene and diplopterol ( 15% of products
formed) were evaluated by a System 2000 Imaging Scanner
(Packard).
Cyclization of oxidosqualene. Purified SHC (3–80 lg) was
incubated at 55 °Cfor30minin1mL0.1
M
Na citrate
buffer (pH 6.0) containing 1.5 mgÆmL
)1
polidocanol and
10 l
M
[
14
C]oxidosqualene (3000 c.p.m.). The reaction was
stopped with KOH and the nonsaponifiable lipids were
extracted as described for squalene. The extract was
chromatographed on silica gel plates developed in CH
2
Cl
2
.
The radioactivities of chromatographic bands (oxidosqua-
lene and hop-22(29)-en-3-ol) [23] were evaluated as des-
cribed for squalene.
Time-dependent inactivation
SHC (0.12–3.2 mgÆmL

The samples were then analysed by 10% SDS/PAGE, the gel
was stained, enhanced with 20% 2,5-diphenyloxazole in
acetic acid, washed in water, dried and exposed to a Kodak
X-OMAT XAR-5 film at )80 °C for 7–10 days.
Digestion and MS analysis of labelled protein
For tryptic proteolysis, labelled protein (1 mg) dissolved in
buffer-B (20 lL, 20 m
M
Tris/HCl, pH 8.0; 0.6% C
8
E
4
;
200 m
M
NaCl) was precipitated with 80 lLEtOHat0°C
to eliminate any detergent present. Precipitated protein was
resuspended in 50 lL buffer-D (10 m
M
N-ethyl-morpho-
line-HOAc, pH 7.8, 2 m
M
CaCl
2
) and digested with 10 lg
trypsin (Promega) at 37 °C and occasional agitation for 2 h.
Trypsin addition and incubation were repeated, the protease
was heat inactivated (5 min at 98 °C) and fragments were
analysed by LC-ESI-MS. Ten lLdigestedSHCwere
applied onto a Phenomenex RP column (Jupiter 5 l

less active than either the wild-type or the single mutant, as
indicated by the values of k
cat
and k
cat
/K
M
.
A radioactive thiol modifying squalene-analogue
[(CNDT-squalene (1)] was first used to test the ability of a
squalenoid molecule to reach the channel constriction.
Native SHC and quadruple mutant (40 lgprotein)were
incubated separately in 0.1
M
Na citrate buffer (pH 6)
containing 1.5 mgÆmL
)1
polidocanol, for 60 min at 55 °C
with 0.2 m
M
[1-
3
H]-CNDT-squalene (1)(10· 10
6
d.p.m.,
5.26 · 10
10
d.p.m.Æmmol
)1
). SDS/PAGE of modified

)1
)
k
cat
/K
M
(min
)1
l
M
)1
)
K
M
(l
M
)
k
cat
(min
)1
)
k
cat
/K
M
(min
)1
l
M

bearing only the cysteine residue of the channel constriction.
DTS (5) was the most effective inactivating agent of the
quadruple mutant, as indicated by t
½
inactivation values
(Table 2 and Fig. 6). Covalent modification of C435 by
DTS (5) was confirmed by tryptic digestion and MS analysis
of the quadruple mutant treated with the inhibitor. The
DTS-labelled tryptic peptide comprising C435 has a theor-
etical molecular mass of 6895.8 Da. Deconvoluted mass
spectra (Fig. 7B) indicated a molecular mass of 6893.0 Da
and refined data analysis a molecular mass of 6894.2 Da for
the expected peptide recorded by scans nos 1481–1486. The
corresponding peak with elution time 47.85 min was the
most prominent peak of the chromatogram (Fig. 7A). A
difference between the theoretical and the experimental
values of 1.6 Da can be tolerated due to the systematic error
of the mass spectrometer (0.01–0.05%). Even if an incom-
plete tryptic digest of SHC with up to two missed cleavages
is taken into account, it can be ruled out that other peptides
caused the observed MS signals, because potential tryptic
peptides are residues 403–466 (one missed cleavage,
7042.6 Da instead of the observed value 6894.2 Da), the
unlabelled peptide containing C435 (6682.2 Da) and resi-
dues 274–332 (two missed cleavages, 6500.4 Da). Further-
more, due to the high intensity of the chromatogram peak
assigned to the labelled peptide it is unlikely that peptides
originating from unspecific cleavage of the labelled SHC
and having a molecular mass close to that of the labelled
peptide contributed to the deconvolution peak. Therefore

C25S/C50S/C455S/C537S by DTS (5). Data are shown for DTS (5)
concentrations of 100 l
M
(r)and20l
M
(j).
Fig. 7. LC-ESI-MS results: RP-HPLC separation of tryptic digested
mutant C25S/C50S/C455S/C537S labelled with DTS (5). Elution time
[min] is denoted above the peaks (A). Deconvoluted spectra of ESI-MS
scans nos 1481–1486 corresponding to the elution time 47.85 min (B).
Ó FEBS 2002 Effects of SH-modifying agents on squalene cyclase (Eur. J. Biochem. 269) 2113
complex interaction between substrate and enzyme. This
approach was successfully adopted with yeast oxidosqua-
lene cyclase, which was irreversibly inhibited by CNDT-
squalene (1), a thiol-reacting squalene-like molecule [24].
SHCs, unlike OSCs, bear no cysteine residues at the active
centre, as they lack the cysteine residue present in the
conserved active site motif DCTA of eukaryotic OSCs (in
prokaryotic cyclases the motif is DDTA). No other cysteine
residues appear at the active centre cavity of SHC. The goal
of binding a squalene analogue inside the active site of SHC
covalently may thus be pursued by introducing a critical
cysteine residue into the active centre cavity by site-directed
mutagenesis, able to serve as a Ôsticky pointÕ for squalene
analogues with thiol-modifying activity. The D376C/C435S
mutant, already used in crystallization experiments of SHC
[5], seemed to fit the above purpose. In this mutant, the
D376 at the reaction initiation site of the central cavity has
been replaced by a cysteine residue, and C435 at the channel
constriction has been replaced by serine. The latter substi-

bearing a cysteine residue at the active site.
DISCUSSION
A series of thiol modifying agents were used as tools to
elucidate some structural/functional features of squalene-
hopene cyclase. The study started from the observation that
C435 of the enzyme, is located on the putative path of the
substrate from the membrane interior to the active centre.
C435 is in fact located at a constriction formed by four
amino-acid residues, which separates the large central cavity
containing residues critical for catalysis from a lipophilic
channel open towards the inner surface of the protein
(Fig. 2). This constriction appears to be sufficiently mobile
to act as a gate for substrate passage, due to the flexibility of
a loop bearing two of the four amino-acid residues as
indicated by the higher crystallographic B-factors [5]. The
ability of the inhibitor CNDT-squalene (1)tomodifyC435
covalently provided the first evidence that a substrate
analogue can move along the lipophilic channel and reach
the enzyme’s putative gate-constriction, establishing that
this is in fact the entrance to the active centre.
Even stronger support for the position of the entrance
gate came from the inactivating experiments with the C25S/
C50S/C455S/C537S mutant, which bears C435 as the only
cysteine of the protein. When this SHC-mutant was exposed
to thiol-modifying agents, especially to the inhibitor DTS
(5), rapid time-dependent inhibition was observed (Fig. 6).
Such inhibition can be explained as a consequence of an
obstruction of the channel constriction. Other explanations,
such as a direct influence of the inhibitor on the catalytic
process, may be ruled out since C435 is not involved in the

½
of thiol-modifying inhibitors on enzy-
matic activity of mutant C435S and mutant D376C/C435S with
oxidosqualene as substrate. Experimental conditions were as described
in Materials and methods. Data are mean values from three inde-
pendent experiments with a mean deviation of ± 10%.
Inhibitor
Inhibitor
concentration
(l
M
)
t
½
(min)
C435S D376C/C435S
Dodecyl-maleimide (3) 200 > 60 12
Squalene-maleimide (2) 200 > 60 1.5
CPTO (4) 200 > 60 36
DTS (5) 200 45 7.5
2114 P. Milla et al. (Eur. J. Biochem. 269) Ó FEBS 2002
[28]. Recently, the preference for oxidosqualene of such
mutants was confirmed with a SHC mutant bearing the
eukaryotic DCTAEA OSC-motif instead of the DDTAVV
SHC-motif [28]. The exchange of the prokaryotic by the
eukaryotic motif was carried out in [23].
The strong time-dependent inactivation of inhibitors on
the D376C/C435S mutant, and the poor inhibition of the
C435S mutant, indicate that inactivation occurs at C376.
Interestingly, squalene-maleimide (2) [11] which should have

Prof. K. Poralla (Universita
¨
tTu
¨
bingen) for providing wild-type SHC,
D. Kessler and B. Fu
¨
llgrabe for help with mutagenesis and C. Warth
for ESI-MS measurements.
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