The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue C435 was probed by labelling experime
Trang 1Thiol-modifying inhibitors for understanding squalene cyclase function
Paola Milla1, Alexander Lenhart2, Giorgio Grosa3, Franca Viola1, Wilhelm A Weihofen2, Georg E Schulz2 and Gianni Balliano1
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 A Avogadro, Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Novara, Italy
The function of squalene-hopene cyclase from
Alicycloba-cillus acidocaldariuswas studied by labelling critical cysteine
residues of the enzyme, either native or inserted by
site-directed mutagenesis, with different thiol-reacting molecules
The access of the substrate to the active centre cavity through
a nonpolar channel that contains a narrow constriction
harbouring a cysteine residue (C435) was probed by labelling
experiments on both a C435S mutant, lacking C435 of the
channel constriction, and a C25S/C50S/C455S/C537S
mu-tant, bearing C435 as the only cysteine residue Labelling
experiments with tritiated
3-carboxy-4-nitrophenyl-dithio-1,1¢,2-trisnorsqualene (CNDT-squalene) showed that the
cysteine residue at the channel constriction was covalently
modified by the squalene-like inhibitor Time-dependent
inactivation of the C25S/C50S/C455S/C537S mutant by a
number of squalene analogues and other agents with
thiol-modifying activity suggested that thiol-modifying C435 caused the
obstruction of the channel constriction thus blocking access
of the substrate to the active site The tryptic fragment comprising C435 of the quadruple mutant labelled with the most effective inhibitor had the expected altered molecular mass, as determined by LC-ESI-MS measurements The arrangement of the substrate in the active site cavity was studied by using thiol reagents as probes in labelling experiments with the double mutant D376C/C435S in which D376, supposedly the substrate-protonating residue, was substituted by cysteine The inhibitory effect was evaluated
in terms of the reduced ability to cyclize oxidosqualene, as the mutant is unable to catalyse the reaction of squalene to hopene Among the inhibitors tested, the substrate analogue 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-H2 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
A nonpolar channel connects the plateau and the active centre through a narrow constriction formed by four amino-acid residues, which appear to act as a gate that permits substrate passage (Fig 2) The cavity hosting the active site is lined by nonpolar residues, but has a highly polar patch at the top It seems to be shaped so as to bind the substrate in a specific product-like conformation, to trigger cyclization by protonating a terminal double bond,
to assist ring-closures by stabilizing the cationic intermedi-ates and, finally, to deprotonate the hopanyl cation to form hopene or, in a side reaction, to hydroxylate the cation to form hopan-22-ol (diplopterol)
We report here a new approach for studying the access of the substrate to the active site cavity of A acidocaldarius SHC (E.C 5.4.99.x) and its arrangement in it To this aim,
Correspondence to G Balliano, Dipartimento di Scienza e Tecnologia
del Farmaco, Via P Giuria 9, I-10125 Torino, Italy.
Fax: +39 011 6707695, Tel.: +39 011 6707698,
E-mail: gianni.balliano@unito.it
Abbreviations: OSC, oxidosqualene cyclase; SHC, squalene-hopene
cyclase; CNDT-squalene, 3-carboxy-4-nitrophenyl-dithio-1,1¢,
2-trisnorsqualene; U14266A, (U14),
3b-(2-dimethylaminoethoxy)-androst-5-en-17-one; CPTO, 2-(4-chlorophenyl)-D2
-thiazoline-1-oxide; DTS, (dimeric thiolsulfinate), 2(4-chlorobenzamido)
ethane-thiosulfinic acid S-2(4-chlorobenzamido) ethyl ester; NaB 3 H 4 , 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
http://hal9000.cisi.unito.it/wf/DIPARTIMEN/Scienza_e_/index.htm
(Received 1 November 2001, revised 18 February 2002, accepted 25
February 2002)
Trang 2critically 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
M A T E R I A L S A N D M E T H O D S
NMR and MS of chemical products
1H-NMR spectra were recorded on a Jeol EX-400 or Jeol
GX-270, with SiMe4 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 40–60C
Tetrahydrofuran was distilled under sodium benzophenone
ketyl Silica gel was 70–230 mesh Squalene was from
Merck, polyoxyethylene 9 lauryl ether (polidocanol) was
from Sigma-Aldrich, Italy), 2,3-oxidosqualene was prepared
as described in [10] 3-Carboxy-4-nitrophenyl-dithio-1,1¢,2-trisnorsqualene (CNDT-squalene) (1), dodecyl- (3) and squalene-maleimide (2) were synthetized as described else-where [11] U14266A (U14; 3b-(2-dimethylaminoethoxy)-androst-5-en-17-one) [12] was provided by Upjohn Company 2-(4-chlorophenyl)-D2-thiazoline was synthetized
as reported previously [13] m-chloroperbenzoic acid, cystamine dihydrochloride, 4-chlorobenzoyl chloride and triethylamine were purchased from Sigma-Aldrich
2-(4-Chlorophenyl)-D2-thiazoline-1-oxide (CPTO) (4)
To an ice-cold and well stirred solution of 2-(4-chlorophe-nyl)-D2-thiazoline (2 g, 0.0101 mol) in CH2Cl2 (20 mL), m-chloroperbenzoic acid (2.05 g, 0.0101 mol) dissolved in
CH2Cl2(40 mL) was slowly added The mixture was stirred for 3 h on ice while a precipitate appeared CHCl3(60 mL) was then added and the solution was washed with 5% NaHCO3(2· 120 mL) and saturated brine (40 ng NaCl in
100 mL H2O; 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 CHCl3as 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 (CDCl3) d: 3.14 ( m, 1H, 5-Ha), 3.36 (m, 1H, 5-Hb), 4.68 (m, 1H, 4-Ha), 4.91 (m, 1H, 4-Hb), 7.49 (d, 2H, aromatic protons), 8.04 (d, 2H, aromatic protons); IR (KBr)
mmax: 3375, 3040, 2980, 2920, 1612, 1590, 1485, 1400Æcm)1;
UV (CH3OH) kmax: 203, 260
N,N¢-(Dithiodi-2,1-ethanediyl)bis 4-chlorobenzamide To
an ice-cold and well stirred suspension of cystamine dihydrochloride (0.77 g, 0.00343 mol) and triethylamine (12 mL, great excess) in CHCl3(10 mL), 4-chlorobenzoyl chloride (1.5 g, 0.00857 mol) dissolved in chloroform (4 mL) was slowly added under argon atmosphere The mixture was stirred overnight at room temperature CHCl3 (10 mL) was then added and the mixture was extracted with chloroform (2· 50 mL) and chloroform/ethyl acetate 50/
50 (1· 50 mL) The pooled organic phases were washed with 5% NaHCO3 (2· 50 mL) and saturated brine (1· 50 mL) After anhydrification with anhydrous sodium 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);1H-NMR (CDCl3/CD3OD) d: 2.81 ( t, 4H, -CH2 -S), 3.57 (t, 4H, -CH2-N), 7.23 (d, 4H, aromatic protons), 7.61 (d, 4H, aromatic protons); IR (KBr) mmax: 3302, 3236,
1638, 1628, 1597, 1541, 1489Æcm)1; UV ( CH3OH) kmax: 204, 235
2(4-Chlorobenzamido) ethanethiosulfinic acid S-2(4-chlorobenzamido) ethyl ester (DTS ¼ dimeric thiolsulf-inate) (5) A solution of N,N¢-(dithiodi-2,1-ethanediyl)bis 4-chlorobenzamide (400 mg, 0.932 mmol) dissolved in
CH2Cl2(25 mL) was stirred while m-chloroperbenzoic acid (85% purity, 147 mg, 0.932 mmol) was slowly added; it was then allowed to react for a further 3 h with continuous stirring CH2Cl2(20 mL) was then added and the mixture was washed with 5% NaHCO3(2· 30 mL) and saturated brine (1· 50 mL) After anhydrification with anhydrous
Fig 1 Reactions catalysed by (A) prokaryotic SHCand (B) eukaryotic
OSC.
Fig 2 Surface representation of SHCsliced in the middle of the
molecule with nonpolar (yellow), positively charged (blue) and negatively
charged (red) areas The large internal cavity is connected with the
hydrophobic plateau on the right by a nonpolar channel A detergent
molecule LDAO that has been found in a crystal structure of SHC [4]
is shown as ball-and-stick model The catalytic acid D376 and the
residues forming the channel constriction are indicated (V174 lying in
front of C435 was omitted for clarity) The figure was produced with
GRASP [9].
Trang 3sodium sulfate the solvent was evaporated under reduced
pressure The crude product was purified by column
chromatography on silica with CHCl3 and then CHCl3/
CH3OH 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);1H-NMR (CD3OD) d: 3.57 ( m, 4H, -CH2
-SO and -CH2-S), 3.82–3.93 (m, 4H, -CH2-N), 7.53 (m, 4H,
aromatic protons), 7.88 (m, 4H, aromatic protons); IR
(KBr) mmax: 3680, 3412, 2920, 1664, 1597, 1541, 1480Æcm)1;
UV (CH3OH) kmax: 203, 236
Radiochemicals
[2-14C]-mevalonate (50 mCiÆmmol)1) was from NEN [14
C]-squalene and [14C]-3S-2,3-oxidosqualene were prepared by
incubating an S10supernatant (25 mg proteins) of a pig liver
homogenate with 1 lCi [14C]-mevalonate in the presence of
the OSC inhibitor U14266A (U14) [12], essentially as
described by Popjak [14] The nonsaponifiable lipids were
separated by two-step TLC on silica gel plates (Merck)
(20· 20 cm, 0.5 mm layer) The plates were first developed
in light petroleum to a height of about 10 cm above the
origin After drying, the plates were developed to 15 cm
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 N2 and [14C]-squalene and
[14C]-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 [1-3H]trisnorsqualene
thioacetate; (iii) synthesis of [1-3H]trisnorsqualene thiol;
(iv) transformation of [1-3H]trisnorsqualene thiol into [3
H]-CNDT-squalene (1)
(i) [1-3H]Trisnorsqualene alcohol: [1-3H]-(4E,8E,12E, 16E)-4,8,13,17,21-pentamethyl-4,8,12,16,20-docosapentaen-1-ol Pure trisnorsqualene aldehyde, obtained as described
by Ceruti et al [17] (20.5 mg, 0.053 mmol) was dissolved in methanol (0.5 mL) and added to the NaB3H4-containing phial (total activity 25.0 mCi, specific activity 500 mCiÆ mmol)1, total amount 0.05 mmol) After 3 h, NaBH4 (excess, 6.0 mg, 0.159 mmol) was added to complete the reaction After an additional hour, the methanol was evaporated under nitrogen and the reaction mixture was dissolved in dichloromethane, transferred to a single necked flask, and evaporated to dryness under reduced pressure to give crude [1-3H]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 [1-3H]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) [1-3H]Trisnorsqualene thioacetate: [1-3H]-(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 0C The mixture was stirred at
0C for 30 min and produced a white precipitate A solu-tion of [1-3H] trisnorsqualene alcohol (18 mg, 0.046 mmol) and thiolacetic acid (37.2 mg, 0.488 mmol) in 0.5 mL anhydrous tetrahydrofuran was added dropwise under nitrogen and the mixture stirred for 1 h at 0C and 1 h
at room temperature The mixture was evaporated under nitrogen and the crude product triturated in light petroleum; the suspension was purified by column chromatography on silica gel with 100% light petroleum to remove impurities, then light petroleum/diethylether 99.5 : 0.5 to give pure [1-3H] trisnorsqualene thioacetate (16.5 mg, 0.037 mmol) The radiochemical purity of the thioacetate was determined
by radiochromatogram with light petroleum/diethylether
98 : 2 and then revealed with iodine vapour Total activity: 3.6 mCi; specific activity: 92 mCi mmol)1; chemical yield: 80%
(iii) [1-3H]Trisnorsqualene thiol: [1-3H]-(4E,8E,12E,16E) 4,8,13,17,21-pentamethyl-4,8,12,16,20-docosapentaenyl 1-thiol [1-3H]trisnorsqualene thioacetate (16.5 mg, 0.037 mmol) was dissolved in anhydrous diethylether (2 mL) and added dropwise to a suspension of LiAlH4 (11.6 mg, 0.31 mmol) in anhydrous diethylether (5 mL) under nitrogen The mixture was stirred 25 min at room temperature then 25 min under reflux LiAlH4 excess was destroyed by the careful addition of 7 mL 1MHCl 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) [1-3H]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 [1-3H] CNDT-squalene The asterisk
indicates position of 3 H-label: (i) NaB 3 H 4 (ii) (Ph) 3 P, CH 3 COSH,
diisopropyl azodicarboxylate; (iii) LiAlH 4 (iv)
5,5¢-dithiobis(2-nitro-benzoic acid).
Trang 4in ethanol (9 mL) To this solution [1-3H] trisnorsqualene
thiol dissolved in ethanol (10 mL) was added under
nitrogen The mixture was stirred overnight at room
temperature and then the solvent evaporated under reduced
pressure The resulting yellow oil was purified by column
chromatography on silica gel with
dichloromethane/meth-anol 92 : 8 as eluent to give [1-3H] trisnorsqualene
nitro-benzoic acid (1) (11.1 mg, 0.0185 mmol) The radiochemical
purity of the disulfide was determined by
radiochromato-gram with dichloromethane/methanol 85 : 15 and then
revealed with iodine vapour Total activity: 0.91 mCi;
specific activity: 92 mCiÆmmol)1; chemical yield: 50%
Expression and purification of recombinant SHC
Wild-type SHC was kindly provided by Prof K Poralla
(Universita¨t Tu¨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-torTM, 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 megaprimers
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¢-CTCCTCTCCAGCCAAAAGG-3¢;
ApaI, 5¢-GTACAGGGCCCACGTGCCG-3¢;
TG-3¢; MP-HindIII, 5¢-CAGCCAAGCTTGCATGCCTG
-3¢; MP-SacI, 5¢-CATGCAGAGCTCGAAC GGCG-3¢
The purified PCR products were digested with EcoRI/
ApaI and SacI/HindIII, respectively, purified by agarose gel
electrophoresis and gel extraction, and were ligated into the
vector fragments obtained in an analogous manner Ligation
products were transformed into Escherichia coli JM105 cells
according to standard protocols [22] Mutagenesis results
were validated by DNA sequencing (SeqLab, Go¨ttingen,
Germany) Expression and purification of the mutant SHC
was performed as described by Wendt et al [19]
Enzyme assay
Cyclization of squalene Purified SHC (3–5 lg) was
incubated at 55C for 30 min in 1 mL 0.1M Na citrate
buffer (pH 6.0) containing 1.5 mgÆmL)1 polidocanol and
10 lM [14C]squalene (3000 c.p.m.) The reaction was
stopped by adding 1 mL 10% KOH in MeOH, and the
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 55C for 30 min in 1 mL 0.1M Na citrate buffer (pH 6.0) containing 1.5 mgÆmL)1 polidocanol and
10 lM[14C]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 CH2Cl2 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)1) was preincubated at 55C in 0.1M
Na citrate buffer (pH 6.0) containing 1.5 mgÆmL)1 polido-canol and different concentrations of reagents After the preincubation period, the inhibitor concentration was de-creased by 40-fold dilution, and the squalene or oxidosqua-lene cyclizing activity was determined as described above
Labelling with radioactive CNDT-squalene (1) SHC wild-type and mutants (40 lg) were incubated at 55C for 1 h in 0.1M Na citrate buffer (pH 6.0) containing 1.5 mgÆmL)1polidocanol and 2 mM [3H]-CNDT-squalene (1) ( 10· 106d.p.m., 5.26· 1010d.p.m./mmol) The enzyme was precipitated with 5 vol cold acetone The precipitate was washed twice with cold acetone and resuspended in sample buffer for SDS/PAGE with or without 2-mercaptoethanol 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 mM Tris/HCl, pH 8.0; 0.6% C8E4;
200 mMNaCl) was precipitated with 80 lL EtOH at 0C
to eliminate any detergent present Precipitated protein was resuspended in 50 lL buffer-D (10 mM N-ethyl-morpho-line-HOAc, pH 7.8, 2 mMCaCl2) and digested with 10 lg trypsin (Promega) at 37C and occasional agitation for 2 h Trypsin addition and incubation were repeated, the protease was heat inactivated (5 min at 98C) and fragments were analysed by LC-ESI-MS Ten lL digested SHC were applied onto a Phenomenex RP column (Jupiter 5 l C4300 A, 150 mm· 2.0 mm), the fragments were eluted in
a gradient 0–50% CH3CN in 0.1% formic acid and mapped
by ESI-MS (Finnigan TSQ7000, scanzone 200–2300 amu/z) Deconvolution and data analysis were performed with the programBIOWORKS8.2 (Finnigan)
R E S U L T S
Blocking substrate access to the active centre The idea of inhibiting the enzyme by blocking substrate access to the active centre stemmed from the observation
Trang 5that one of the four amino-acid residues present at the
constriction of the hypothetical access channel is a cysteine
(C435), an amino-acid residue that is easy to modify
(Fig 2) The possible inhibitory effect of labelling the C435
residue with a series of substrate analogues or other thiol
modifying agents ( Fig 4) was then studied As SHC
possesses five cysteine residues and no disulfide bridges,
labelling experiments were performed not only on the native
protein but also on two mutants obtained by site-directed
mutagenesis: C435S, lacking C435 at the channel
constric-tion, and the C25S/C50S/C455S/C537S-mutant (quadruple
mutant), bearing C435 as its only cysteine residue The
mutants were first characterized for their optimal
tempera-ture and kinetic parameters (Table 1) All of the mutants
showed the optimal temperature at 60C like wild-type
SHC (data not shown) The quadruple mutant proved to be
less active than either the wild-type or the single mutant, as
indicated by the values of kcatand kcat/KM
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 lg protein) were incubated separately in 0.1M Na citrate buffer (pH 6) containing 1.5 mgÆmL)1polidocanol, for 60 min at 55C with 0.2 mM[1-3H]-CNDT-squalene (1) ( 10· 106 d.p.m., 5.26· 1010 d.p.m.Æmmol)1) SDS/PAGE of modified enzyme followed by fluorography were then used to verify labelling of the protein by the inhibitor An aliquot of incubated protein was treated with 2-mercaptoethanol followed by electrophoretic analysis to verify formation of disulfide bridges between thiol residues of the protein and the squalenoid radioactive moiety of the inhibitor
As shown by the results reported in Fig 5, both wild-type protein and mutant were covalently labelled by the radio-active inhibitor The unambiguous labelling of the quad-ruple mutant, bearing only C435, indicates that inhibitor has reached and modified the cysteine residue at the channel constriction Inactivation experiments carried out at con-centrations of inhibitors up to 0.5 mMshowed that CNDT-squalene (1) behaves as a poorly irreversible inhibitor of the quadruple mutant
Fig 4 Structures of synthesized inhibitors.
Table 1 Kinetic parameters for the wild-type and mutant SHCs with squalene or oxidosqualene as substrates The initial rates were measured at 55 C
as described in Material and methods The kinetic values were determined from double-reciprocal plots NA, not applicable.
Protein
Substrate: squalene Substrate: oxidosqualene
K M
(l M )
k cat
(min)1)
k cat /K M
(min)1l M )1 )
K M
(l M )
k cat
(min)1)
k cat /K M
(min)1l M )1 )
C25S/C50S/C455S/C537S 13.6 ± 5.4 0.39 ± 0.16 0.03 1.0 ± 0.5 0.042 ± 0.01 0.042
Fig 5 SDS/PAGE and fluorography of SHCwild-type and quadruple mutant incubated with [1- 3 H] CNDT-squalene (1) Coomassie stained SDS/PAGE (lanes 1 and 2) and fluorography (lanes 3–6) Samples without 2-mercaptoethanol treatment (lanes 3 and 5) and with 2-mercaptoethanol treatment (lanes 4 and 6) M, markers.
Trang 6The effect of obstructing the channel constriction bearing
C435 was then evaluated by treating the quadruple mutant,
having C435 as the only thiol residue, with other
thiol-modifying agents of different shapes and sizes (Fig 4)
Some of them [squalene-maleimide (2) and
dodecyl-malei-mide (3)] were designed to link a cysteine residue with a
maleimide ring bearing a flexible lipophilic chain, others
[CPTO (4) and DTS (5)] to modify the thiol residue with an
arm bearing the bulky and rigid chlorophenylketone group
at its ends The effect of the inhibitors on the quadruple
mutant was compared with that observed on the C435S
mutant, lacking only the cysteine residue of the channel
constriction (Table 2)
Inhibitors proved to have little effect on the mutant
lacking the cysteine at the channel constriction, whereas
they were all good inactivating agents for the mutant
bearing only the cysteine residue of the channel constriction
DTS (5) was the most effective inactivating agent of the
quadruple mutant, as indicated by t½inactivation 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 the identification of the labelled peptide could be interpreted
as unambiguous
Binding of a squalene analogue inside the active centre Labelling a specific amino-acid residue at the active centre with a substrate analogue is a crucial step in studying the
Table 2 Inactivating effect t ½ of thiol-modifying inhibitors on
enzy-matic activity of mutant C435S and quadruple mutant C25S/C50S/
C455S/C537S with squalene as substrate Experimental conditions
were as described in Materials and methods Data are mean values
from three independent experiments with a mean deviation of ± 10%.
Inhibitor
Inhibitor concentration (l M )
t ½ (min)
C435S
C25S/C50S/
C455S/C537S Dodecyl-maleimide (3) 500 > 60 30
Squalene-maleimide (2) 200 > 60 45–60
Fig 6 Half-logarithmic plot of time-dependent inactivation of mutant
C25S/C50S/C455S/C537S by DTS (5) Data are shown for DTS (5)
concentrations of 100 l (r) and 20 l (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).
Trang 7complex 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 CNDT-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 sticky point 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-tution was required to allow thiol-reacting molecules to
move to their target located in the active site cavity, without
interacting with the cysteine residue at the channel
constric-tion
A series of thiol-reacting agents were tested as
time-dependent inhibitors of the D376C/C435S mutant The
SHC mutant C435S was used as control Mutant D376C/
C435S lost the ability to cyclize squalene due to the absence
of one of the two aspartate residues in the crucial DDTA
motif [25], but it still shared the ability to cyclize
oxido-squalene with wild-type SHC [23] and with the control
C435S mutant Therefore, the inhibitory properties of thiol
reagents were assayed towards the oxidosqualene cyclizing
activity First, enzymatic activity of the mutants with
oxidosqualene as a substrate, was characterized and
compared with wild-type enzyme Both the D376C/C435S
and the C435S mutants showed the same temperature
profile (data not shown) for oxidosqualene cyclizing
activ-ity, with maximum activity at 60C, coincident with the
optimal temperature for squalene cyclization by wild-type
SHC [3] Comparison of kinetic parameters showed that the
double mutant was less efficient in cyclizing oxidosqualene
than either the wild-type or the mutants bearing the native
DDTA motif (Table 1)
All thiol-reacting agents tested had a stronger inhibitory
effect on the D376C/C435S mutant, which was
time-dependent, than they did on the C435S mutant with intact
initiation site motif DDTA (Table 3) The reactive
sub-strate-analogue squalene-maleimide appeared to be the most effective inhibitor of the D376C/C435S mutant bearing a cysteine residue at the active site
D I S C U S S I O N
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) to modify C435 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 reaction mechanism as shown by the essentially unchanged activity of the C435S mutant compared to wild-type (Table 1) Moreover, when the inhibitors could in principle overcome the barrier of the channel constriction and act directly in the active site cavity, as in the case of the C435S mutant, poor inhibition effect was observed (Table 2) Interestingly, the inhibitors squalene- and dodecyl-malei-mide, which modify thiol residues with a group bearing a mobile and flexible chain, proved to be less active than CPTO (4) and DTS (5) The reason for the different effectiveness of the inhibitors could, of course, simply result from their different reactivity or, as in the case of DTS (5), from some enhancement effect due to the formation of reactive intermediates during the reaction between the intact inhibitor and thiol groups [26,27] Moreover, a difference depending on size and rigidness of groups modifying C435 may occur
The series of thiol-reacting agents used to study access of the substrate to the active site of SHC was also found to be a useful tool for studying the possibility of forming an enzyme–inhibitor complex with the inhibitor covalently bound inside the active centre cavity The mutant used for the study was the D376C/C435S mutant bearing a cysteine residue at the reaction initiation site in the central cavity and
a serine substituting the cysteine at the channel constriction This mutant is unable to cyclize squalene [25], but recognizes 2,3-oxidosqualene as a substrate, an ability shared with wild-type SHC and with mutants bearing at least one of the two aspartate residues of the conserved sequence DDTA
Table 3 Inactivating effect t 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
Trang 8[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
a higher ability to deliver a reactive group to the
squalene-hosting cavity of the enzyme, proved to be the most
powerful inactivating agent It appears possible that, for the
first time, a covalent SHC–inhibitor complex has been
formed, in which a squalene analogue in a noncyclized form
is bound to the squalene-hosting cavity covalently These
results open the fascinating prospect of solving the structure
of a SHC with a complexed squalenoid molecule, and
determining in detail the interactions between residues at the
active site and the squalene skeleton in its not-yet cyclized
conformation
In summary, rationally designed mutants of SHC, C25S/
C50S/C455S/C537S (quadruple mutant) and D376C/
C435S, obtained by site-directed mutagenesis, have been
effectively inhibited by thiol-reacting molecules DTS (5) and
squalene-maleimide (2) in a time-dependent manner
Ex-perimental evidence suggests that DTS (5) inhibits the
quadruple mutant by obstructing the substrate access to the
active site, while squalene-maleimide (2) inactivates the
D376C/C435S mutant by irreversible occupation of the
active centre cavity These results, in particular those
relating to the D376C/C435S mutant, may be regarded as
the first step towards the goal of preparing stable covalent
complexes for structural analysis
A C K N O W L E D G E M E N T S
This work was supported by the Ministero dell’Universita` e della
Ricerca Scientifica e Tecnologica (MURST), Italy (ex 60%), and by the
Deutsche Forschungsgemeinschaft under SFB-388 The authors thank
Prof K Poralla (Universita¨t Tu¨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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