Escherichia coli cyclopropane fatty acid synthaseMechanistic and site-directed mutagenetic studies Fabienne Courtois, Christine Gue´rard, Xavier Thomas and Olivier Ploux Laboratoire de C
Trang 1Escherichia coli cyclopropane fatty acid synthase
Mechanistic and site-directed mutagenetic studies
Fabienne Courtois, Christine Gue´rard, Xavier Thomas and Olivier Ploux
Laboratoire de Chimie Organique Biologique, UMR7613 CNRS, Universite´ Pierre et Marie Curie, Paris, France
Escherichia colifatty acid cyclopropane synthase (CFAS)
was overproduced and purified as a His6-tagged protein
This recombinant enzyme is as active as the native enzyme
with a Kmof 90 lMfor S-AdoMet and a specific activity of
5· 10)2lmolÆmin)1Æmg)1 The enzyme is devoid of organic
or metal cofactors and is unable to catalyze the wash-out of
the methyl protons of S-AdoMet to the solvent, data that do
not support the ylide mechanism Inactivation of the enzyme
by 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), a pseudo
first-order process with a rate constant of 1.2M )1Æs)1, is not
protected by substrates Graphical analysis of the
inactiva-tion by DTNB revealed that only one cysteine is responsible
for the inactivation of the enzyme The three strictly
con-served Cys residues among cyclopropane synthases, C139,
C176 and C354 of the E coli enzyme, were mutated to
serine The relative catalytic efficiency of the mutants were
16% for C139S, 150% for C176S and 63% for C354S The three mutants were inactivated by DTNB at a rate com-parable to the rate of inactivation of the His6-tagged wild-type enzyme, indicating that the Cys responsible for the loss
of activity is not one of the conserved residues Therefore, none of the conserved Cys residues is essential for catalysis and cannot be involved in covalent catalysis or general base catalysis The inactivation is probably the result of steric hindrance, a phenomenon irrelevant to catalysis It is very likely that E coli CFAS operates via a carbocation mechanism, but the base and nucleophile remain to be identified
Keywords: cyclopropane fatty acid synthase; hydrogen isotope exchange; enzymatic reaction mechanism; site-directed mutagenesis; chemical modification
Cyclopropane synthases constitute an interesting class of
enzymes that catalyze the cyclopropanation of unsaturated
lipids in bacteria [1], plants [2,3] and parasites [4]
Escheris-hia colicyclopropane fatty acid synthase (CFAS) [5–9] and
its closely related homologs from Mycobacterium
tuber-culosis[10] are the best known representatives of this class
of enzymes In E coli, cyclopropanation is thought to be
involved in long-term survival of nongrowing cells and is
often associated with enviromental stresses [1] In M
tuber-culosis, cyclopropanation has recently been associated
with virulence and persistance of the pathogen [11] Hence,
cyclopropane synthases might be good targets for new
antituberculous drugs Indeed, tuberculosis remains a major cause of death in the world and there is a real need for new drugs to combat strains of M tuberculosis that are resistant
to existing drugs [12] We have been interested in studying CFAS from E coli as a model for M tuberculosis cyclo-propane synthases, for which an in vitro assay is still lacking Our goal is to contribute to the elucidation of this intrigu-ing enzymatic reaction, but also to discover inhibitors of cyclopropane synthases that might be good leads to antituberculous drugs [13]
This enzymatic cyclopropanation reaction proceeds by transfer of a methylene group from the activated methyl group of S-adenosyl-L-methionine (S-AdoMet) to the (Z)-double bond of an unsaturated fatty acid chain, resulting in the formation of a cyclopropane ring on the alkyl chain (Scheme 1) Early in vivo studies [14–16] showed that two of the three methyl protons of S-AdoMet are retained in the product, although some exchange was observed under certain conditions [17,18], and that the vinylic and allylic protons of the substrate are also retained
in the product The stereochemistry is also retained; that is, the (Z)-double bond gives a cis-cyclopropane [5], although trans-cyclopropanes are also found in M tuberculosis mycolic acids [10] Chiral methyl analysis was also conduc-ted in vivo using Lactobacillus plantarum cells, and showed retention of the stereochemistry of the reaction [19] This experimental observation is not in favor of a carbenoid species (see below), which would probably racemize Two types of reaction mechanism have been proposed for this fascinating reaction: a carbocation type and an ylide type mechanism, schematically represented in Scheme 2
Biologique – UMR CNRS 7613, Boıˆte 182, Tour 44–45, 4 Place
Jussieu, F-75252 Paris cedex 05, France Fax: +33 1 44 27 71 50,
Tel.: +33 1 44 27 55 11, E-mail: ploux@ccr.jussieu.fr
Abbreviations: BSA, bovine serum albumin; CFAS, cyclopropane
fatty acid synthase; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid);
EDTA, ethylenediaminetetraacetic acid; NEM, N-ethylmaleimide;
S-adenosylhomo-cysteine.
-methionine:unsaturated-phospholipid methyltransferase (cyclizing),
cyclopropane-fatty-acyl-phospholipid synthase, cyclopropane
synthase (EC 2.1.1.79).
Note: A website is available at http://www.ccr.jussieu.fr/umr7613/
(Received 28 July 2004, revised 16 September 2004,
accepted 18 October 2004)
Trang 2Even though the mechanism involving a carbocation
intermediate is often cited in the literature [1,10,20], the
other reasonable alternatives deserve consideration and in
particular the metal-assisted ylide mechanism [21] However,
recent crystallographic data [22], inhibition and mechanistic
studies [13,18,23,24], and data reported in this study argue
in favor of the carbocation mechanism On the basis of
chemical modification experiments [9], the involvement of a
cysteine residue in the catalysis has been invoked Indeed, the
thiolate side chain could be either the base that is required for
abstraction of the methyl proton, or could stabilize the
carbocation, if that intermediate were formed, or even
participate in a covalent catalysis (Scheme 2) Interestingly,
the three dimensional structure of three cyclopropane
synthases from M tuberculosis [22] showed the presence of
two cyseines at, or near, the active site: C139 and C354
(E coli CFAS numbering) Futhermore, these residues, as
well as C176 (E coli numbering), are strictly conserved in all
cyclopropane synthases discovered so far [1]
We report here the purification of a His6-tagged CFAS
and its characterization We also report exchange
experi-ments that are not in favor of the ylide mechanism The role
of the conserved cysteines was studied using chemical
modification and site-directed mutagenesis It was found
that the cysteines are not essential for catalysis
Experimental procedures
General
E colistrains JM109 and BL21(DE3) were from Promega (Madison, WI, USA), and E coli K12 was obtained from the Institut Pasteur Collection (CIP; Paris, France) Plasmid pET-24(+) was obtained from Novagen (Darmstadt, Germany) Synthetic oligonucleotides were products of Proligo (Paris, France) and were used without any fur-ther purification Chemicals were purchased from Sigma-Aldrich (Saint Quentin, France) and were of the highest purity available S-[Methyl-14C]adenosyl-L-methionine (60 mCiÆmmol)1) and S-[methyl-3H]adenosyl-L-methionine (85 CiÆmmol)1or 15 CiÆmmol)1) were from New England Nuclear (Boston, MA, USA) Restriction enzymes, Taq polymerase, T4 DNA ligase and molecular biology kits were either from Promega or from Roche (Meylan, France) Culture medium components were purchased from Difco Laboratories (Detroit, MI, USA) Chromatographic equip-ment (GradiFrac) and column phases were from Amersham Biosciences (Orsay, France) UV-visible spectra were obtained on an Uvikon-930 Kontron spectrophotometer (Munchen, Germany) or a Lambda-40 Perkin Elmer apparatus (Norwalk, CT, USA) Scintillation counting was run on a 1214 Rackbeta LKB Wallac radioactivity counter (Perkin Elmer) Sonication was performed on a VibraCell sonicator from Bioblock (Illkirch, France) SDS/ PAGE was run on a Bio-Rad Protean II system (Hercules,
CA, USA), using the conditions described by the manufac-turer, and DNA electrophoresis on a Mupid apparatus (Eurogentec, Seraing, Belgium), in 40 mM Tris/acetate buffer, pH 7.5, 1 mMEDTA Centrifugations were run on
a Sorval RF5plus centrifuge (DuPont, Kendro, Cortaboeuf, France).1H and13C-NMR spectra were obtained on an AC
400 MHz Bruker apparatus (Rheinstetten, Germany) Plasmid construction and site-directed mutagenesis The wild-type histidine-tagged CFAS recombinant gene was obtained using PCR amplification of the cfa gene from
E coli K12 genomic DNA Briefly, E coli K12 genomic DNA was purified using the Wizard Genomic kit from Promega, and the cfa gene was amplified using Taq DNA polymerase (Promega) and the following two primers: 5¢-CGCGAATTCAGGAGGATTTTATGCACCACCA CCACCACCACAGTTCATCGTGTATAGAAGAA-3¢ containing an EcoRI site, a ribosome binding site and a His6-tag sequence, and 5¢-CGCAAGCTTTTAGCGAGC CACTCGAAG-3¢ containing a HindIII site The DNA fragments was purified (PCR Preps, Promega), digested by EcoRI and HindIII, purified on agarose gel and ligated into pET-24(+) previously cut by the same restriction enzymes After transformation in E coli JM109, positive clones were selected and the plasmid extracted and purified (Wizard Plus Minipreps, Promega) for DNA sequencing (ECSG, Evry, France) Plasmid pET-24H6cfa, thus obtained, was used for transformation in E coli BL21(DE3) and this construction afforded efficient expression of the enzyme The mutated cfa genes, cfaC139S, cfaC176S and cfaC354S (numbering corresponds to the wild-type sequence, that
is without counting the N-terminal His-tag that has
R 1 S R 2
CH 3
CH 3
CH 3
Enz-Nu
H H
C H H H
R 1
S R 2
H 3 C
Ylide
R 1
S R 2
CH 2
OR
A
B
Enz Metal
Metal
CH 2
Enz–Nu Enz–Base
Enz–Base
Carbenoid
H H
Scheme 2 Plausible reaction mechanisms for the catalyzed
cyclopro-panation (A) The carbocation mechanism; (B) the ylide mechanism.
This mechanism would most probably require a metalloenzyme and
transfer of a carbenoid to the metal (details are not shown for clarity).
H H H
H
O A
OH HO
S
NH 3
OOC
CH 3
O A
OH HO S
NH 3
OOC
CFAS
S-Adenosyl-L-methionine S-Adenosyl-L-homocysteine
+ H
Scheme 1 Reaction catalyzed by the cyclopropane synthases In E coli
CFAS the lipid substrate is an unsaturated phospholipid, while in
acyl carrier protein.
Trang 3been engineered) of E coli CFAS were constructed
using the QuikChange Site-Directed Mutagenesis Kit from
Stratagene (La Jolla, CA, USA) The following sets of
mutated primers (mutations are underlined) were used:
C139S: 5¢-CATGCAATATTCCAGCGCTTACTGGAA
AG-3¢ and 5¢-CTTTCCAGTAAGCGCTGGAATATTG
CATG-3¢; C176S: 5¢-GGATATTGGCAGCGGCTGGG
GCGGACTGGC-3¢ and 5¢-GCCAGTCCGCCCCAGCC
GCTGCCAATATCC-3¢; C354S: 5¢-CTGAATGCCTCT
GCAGGTGCTTTCCGCGCC-3¢ and 5¢-GGCGCGCGG
AAAGCACCTGCAGAGGCATTCAG-3¢ Plasmid
pET-24H6cfa was used as the template Transformants
were selected and the plasmids were extracted, purified
(Wizard Plus miniprep kit from Promega) and sequenced
(Eurogentec) to ensure integrity and the presence of the
desired mutation In the case of the C354S mutant,
the mutation was confirmed by digestion with PstI as the
mutation introduces a new restriction site Each mutated
plasmid was then transformed into competent E coli
BL21(DE3) for protein expression
Protein assay
Protein concentrations were determined using the
colori-metric assay described by Bradford [25] and supplied by
Bio-Rad
Phospholipids preparation
Unsaturated E coli K12 phospholipids were prepared
according to Cronan [6] and as originally described by
Ames [26] Once purified the phospholipids were stored as a
chloroform solution at )20 C Aqueous solutions of
phospholipids were prepared by evaporating the chloroform
and resuspending the phospholipids in 20 mM potassium
phosphate buffer, pH 7.4 at the desired concentration
( 20 mgÆmL)1) Phospholipids were assayed using the
ferric hydroxamate method, as described previously [27],
and using tripalmitin standards for calibration
Phospho-lipid solutions were sonicated for 30 s for dispersion prior to
use as substrates Cyclopropanated phospholipids were
extracted, using the same protocol, from isopropyl
thio-b-D-galactoside (IPTG)-induced E coli
BL21(DE3)/pET-24H6cfa cells
CFAS purification
An overnight preculture [10 mL Luria–Bertani (LB)
medium, 50 lgÆmL)1 kanamycin] of E coli BL21(DE3)/
pET-24H6cfa was used to inoculate 800 mL of LB medium
supplemented with 50 lgÆmL)1kanamycin The culture was
shaken (180 r.p.m., 37C), and when the absorbance at
600 nm reached a value of 0.7, IPTG was added at a final
concentration of 100 lM The culture was then shaken
overnight at 37C The cells were collected by
centrifuga-tion (4000 g, 15 min), washed (0.1Mpotassium phosphate
buffer, pH 7.4), centrifuged (4000 g, 15 min) and kept at
)20 C until use The cell paste was resuspended in 40 mL
of 20 mM potassium phosphate buffer, pH 7.4, and the
suspension was sonicated on ice (5 min, with 1 min cooling
period every minute) After centrifugation (15 000 g,
20 min), the supernatent was loaded directly on a nickel
affinity column (Chelating Sepharose, Amersham Bio-science; 1.6 cm i.d., 5 cm long, 10 mL) prepared as recom-mended by the manufacturer and equilibrated with buffer A (20 mMpotassium phosphate buffer, pH 7.4, 0.5MNaCl) The column was successively washed with 30 mL of buffer
A and 30 mL of buffer A containing 5% (v/v) of buffer B (20 mMpotassium phosphate buffer, pH 7.4, 0.5MNaCl, 1.0M imidazole) The proteins were eluted by a linear gradient starting from 5% (v/v) of buffer B to 40% (v/v) of buffer B, in buffer A The column was run at a flow rate of
1 mLÆmin)1and 7 mL fractions were collected The pres-ence of proteins was detected using the Bradford assay and the purity of individual fractions was analyzed by SDS/ PAGE Fractions containing pure CFAS were pooled and desalted on PD-10 columns (Amersham Bioscience) equili-brated with buffer A Highly concentrated enzyme solu-tions were obtained by ammonium sulfate precipitation as follows Solid ammonium sulfate was added at 0C to the enzyme solution, up to 40% saturation, and the precipitated protein was recovered by centrifugation (10 min at
12 000 g) The pellet was then dissolved in the minimum volume of 20 mM potassium phosphate buffer, pH 7.4, 50% (v/v) glycerol, and the enzyme solution was stored at )20 C The mutant proteins, which all carry an N-terminal His6-tag, were purified as described for the His6-tagged wild-type enzyme
Biochemical characterization N-terminal protein sequencing of the enzyme, transfered onto a polyvinylidene fluoride membrane, was obtained at the Plateau Technique d’Analyse et de Microsequenc¸age des Prote`ines (Institut Pasteur) For the determination of the metal content, several samples (18 nmol each) of purified CFAS were lyophilized and subjected to metal analysis (Zn, Ni, Co, Fe, Cu) using ICP-AES methodology (Service Central d’Analyse; CNRS, Vernaison, France) The metal content, in each case, represented less than 1%
of what was expected for 1 mol of metal per mol of enzyme
CFAS assay CFAS activity was assayed as described previously [7] with slight modifications The assay consisted in 1.0 mgÆmL)1 phopholipids, 0.5 mgÆmL)1bovine serum albumin (BSA), 10% (v/v) glycerol, 2 mM dithiothreitol (dithiotheithol), 0.75 mM S-AdoMet, either 14C-labelled (specific radioac-tivity of 5.0 mCiÆmmol)1) or3H-labelled (specific radioac-tivity of 1.5 mCiÆmmol)1), 2 lg CFAS, in 20 mMpotassium phosphate buffer, pH 7.4, in a final volume of 100 lL The reaction was initiated by addition of the enzyme and incubated at 37C for 20 min The reaction was stopped by adding 1 mL 10% (v/v) trichloroacetic acid, and the solu-tion was filtered over glass fiber filters (Whatman GF/c, Middlesex, USA; 25 mm) The filters, adapted on a filtration device (Millipore, Billerica, MA, USA; 1225 model), were washed three times with 1 mL 10% (v/v) trichloroacetic acid, three times with 1 mL H2O, oven-dried (60C, 20 min) and finally counted for radioactivity in
5 mL of scintillation cocktail (Optiphase, Wallac) The activity measured under these conditions was linear with
Trang 4time over a period of 20 min and linear with enzyme
concentration up to 0.1 mgÆmL)1 of protein (data not
shown) One unit of CFAS is defined as the amount of
enzyme that transforms 1 lmol of substrate per min The
kinetic parameters of His6-tagged wild-type and mutant
CFAS were determined by measuring the activity
(as described above for the enzyme assay) at different
concentrations of S-AdoMet Data were analyzed using
nonlinear regression analysis run onKALEIDAGRAPH
soft-ware, to fit to Michaelis–Menten kinetics
S-Adenosyl-homocysteine nucleosidase was purified from an
overproducing strain (E coli BL21(DE3)/pEXH6MTAN),
generously given by K Cornell and M Riscoe (VAMC,
Portland, OR, USA), and assayed as described previously
[28]
pH profile
CFAS activity was determined as described above in the
following buffers: 4-morpolinoethanesulfonic acid (pH 5.5–
7.0), 2-(4-(2-hydoxyethyl)-1-piperazine) ethanesulfonic acid
(pH 7.0–8.5) and
3-(tris(hydroxymethyl)methylmino)-1-proanesulfonicacid (pH 7.7–9.5) all at a concentration of
150 mM The pH was adjusted by adding aqueous HCl or
aqueous NaOH The activity vs pH profile was bell-shaped
and the data points were fitted to Eqn (1):
V¼ Vmax=ð1 þ 10pHpKa1þ 10pKa2pHÞ Eqnð1Þ
using a nonlinear regression analysis supported by
KALEIDAGRAPHsoftware (Synergy Software, Reading, PA,
USA)
Exchange experiments
A sample consisting of 2 lg (45 pmol) CFAS, 2 mM
dithiotheithol, 0.5 mgÆmL)1 BSA, 10% (v/v) glycerol,
20 mM potassium phosphate buffer, pH 7.4, and 1 mM
[methyl-3H]S-AdoMet (13 mCiÆmmol)1) was incubated at
37C for 3 h A control sample that did not contain the
enzyme was run at the same time The reaction was stopped
by dilution with 1 mL water and immediate freezing in
liquid nitrogen Water was then lyophilized, recovered and
counted for radioactivity in 4 mL of scintillation liquid For
the incorporation of deuterium from D2O, the experiment
was run directly in the NMR tube (500 lL, total volume)
The sample consised of 2 lg (45 pmol) CFAS, 2 mM
dithiotheithol, 0.5 mgÆmL)1 BSA, 10% (v/v) glycerol,
20 mM potassium phosphate buffer, pD 7.4 (corrected),
and 1 mM S-AdoMet In order to minimize the H2O
concentration the buffer was exchanged in D2O and
lyophilized prior to use A control sample that did not
contain the enzyme was run at the same time The samples
were incubated for 4 h and were analyzed by1H-NMR The
signal at 3.11 p.p.m., which corresponds to the methyl
group of the natural diastereoisomer of S-AdoMet [(S,S)
configuration], was quantified and compared to an
authen-tic sample of commercial S-AdoMet The methyl group of
para-toluenesulfonate, present in the commercial sample of
S-AdoMet, was used as an internal standard No
modifi-cation of the signal was observed A 13C-NMR (1H
decoupled) spectrum was also recorded to see if any
exchange on the methyl group had occured, because deuterium incorporation would shift the signal and would give a scalar coupling
Thiol titration by 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB)
Thiol titrations were run as described by Riddles et al [29] Breifly, for titration in denaturing conditions, 1.9 nmol (84 lg, 2.4 lMfinal concentration) of purified His6-tagged wild-type CFAS were added to a solution (800 lL final volume) containing 6.0M guanidine hydrochloride, 0.31 mM 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), 0.1M potassium phosphate, pH 7.3, 1 mM EDTA at
20C The exposed thiols were titrated by measuring the change in absorbance at 412 nm (e¼ 13 700 cm)1ÆM )1) For titration under native conditions the same protocol was applied except that the guanidine hydrochloride was not added Thiols were titrated by measuring the change in absorbance at 412 nm (e¼ 14 150 cm)1ÆM )1)
Inactivation by DTNB His6-tagged wild-type and mutant CFAS proteins were treated with various concentration of DTNB in 0.1M potassium phosphate, pH 7.3, 1 mM EDTA, at 20C Aliquots of 30 lL were transferred, at different time points,
to a CFAS assay mixture (final volume of 100 lL) containing, 1.0 mgÆmL)1phopholipids, 0.5 mgÆmL)1BSA, 0.76 mM [methyl-3H]S-AdoMet at a final specific radio-activity of 25 mCiÆmmol)1, 5 mM reduced glutathione to quench the inactivation, in 20 mM potassium phosphate buffer, pH 7.4 The mixture was incubated at 37C for
15 min The reaction was stopped by adding 1 mL 10% (w/v) trichloroacetic acid and treated as described above for radioactivity counting
Protection from inactivation by DTNB His6-tagged wild-type CFAS was incubated with 2 mM DTNB in presence of 1 mgÆmL)1 phospholipids or
380 lM S-AdoMet, in 0.1M potassium phosphate,
pH 7.3, at 20C Residual activity was measured as described for the inactivation experiments (see above) Tsou plot
Two identical samples were prepared as follows His6 -tagged wild-type CFAS (1.9 nmol; 84 lg, 2.4 lM final concentration) was added to a solution (800 lL final volume) containing 0.1M potassium phosphate, pH 7.3,
1 mMEDTA, 0.31 mMDTNB Addition of the enzyme was performed at the same time in both samples and the absorbance at 412 nm (e¼ 14 150 cm)1ÆM )1) was followed against time, using one sample The residual activity was followed against time using the second sample Both incubations were run at 20C Determination of residual activity was carried out as described above for the DTNB inactivation experiments Data obtained at the same time points, that is the number of titrated thiols and the residual activity, were used in the graphical representation described
by Tsou [30]
Trang 5Cloning, expression and purification of CFAS
E coliCFAS was expressed as an N-terminal His6-tagged
recombinant protein, in order to simplify the purification
protocol [9] The recombinant gene, containing an
engin-eered ribosome binding site [31] and a His6-tag was
constructed using PCR-based recombinant technology
and cloned into a pET-24(+) vector A C-terminal tagged
protein was also constructed but the protein was expressed
as an insoluble and inactive polypeptide The N-terminal
His6-tagged construct, pET-24H6cfa, whose DNA sequence
was verified, was used throughout this study
Overexpres-sion in E coli BL21 (DE3)/pET-24H6cfa was optimized by
varying the usual parameters, that is IPTG concentration
(from 40 lM to 1 mM), temperature (20C, 30 C and
37C), and incubation time after induction (from 3 h to
15 h) Our best results were obtained using the following
conditions: 100 lMIPTG, 37C and overnight incubation
The His6-tagged CFAS was purified in two steps (Fig 1)
An affinity nickel chromatography was followed by a
necessary desalting step by gel filtration because high
imidazole concentration inhibits the enzyme activity
Start-ing from 0.8 L of culture (100 mg of protein in the crude
extract with a specific activity of 0.9· 10)2UÆmg)1), 5 mg
of pure protein (Fig 1) was obtained with a specific activity
of 5.0· 10)2UÆmg)1, a value comparable to previously
reported data [9,24] The yield of this purification is 28%,
and the purification fold is 5.5 This simple protocol is fast
enough (a few hours in total) to keep this labile enzyme
active The enzyme was stored best in 20 mM phosphate
buffer, pH 7.4 containing 50% (v/v) glycerol, at)20 C
Assay and characterization
The recombinant CFAS was assayed as described by
Cronan and coworkers with slight modifications [7] We
found that addition of 0.5 mgÆmL)1BSA, 2 mM
dithiothei-thol and 10% (v/v) glycerol substantially stabilized the
enzyme activity during the assay Typically, after 60 min incubation the activity of a sample containing the additives was twice over that of a control sample Addition of S-AdoHcy nucleosidase as suggested previously [7] to hydrolyze the product, a competitive inhibitor [6,13], was not necessary in our assay because the concentration of S-AdoHcy reached was too low to cause inhibition The effect of phospholipid concentration was also checked and
we found a biphasic curve as already observed, with a saturation at 1 mgÆmL)1phospholipid [6] Using this assay
we measured a Kmof 90 ± 5 lMfor S-AdoMet and a kcat
of 2.2 ± 0.1 min)1, values in close agreement to those reported for the native enzyme [7] Therefore, the presence
of the His6-tag does not perturb the catalytic activity N-terminal sequencing showed no contaminants and was
in agreement with the predicted sequence The UV-visible spectrum of the protein did not show any absorption over
300 nm and thus no organic cofactor could be detected Search for usual metals found in proteins (Zn, Ni, Cu, Co, Fe) was unsuccessful We therefore concluded that CFAS has no cofactor, a result in agreement with the three dimensional structure obtained for the M tuberculosis cyclopropane synthases [22]
The effect of pH on the activity of CFAS, under saturating conditions, is shown in Fig 2 The profile is bell-shaped with
a maximum around pH 7.5 Fitting the data to a simple model using Eqn (1) (with two ionisable groups involved in catalysis) gave a pKa1of 6.8 and a pKa2of 8.7
Exchange experiments Exchange of the methyl proton of S-AdoMet catalyzed by CFAS was tested by measuring the wash-out of tritium from the methyl group to the solvent water Incubation of
1
1 0 100
pH
pK
activity was measured at different pH values, using a series of buffers (see Experiental procedures) Each data point represents the average of two independent experiments with less than 5% deviation to the mean.
Ordinates are plotted on a log scale.
and E coli CFAS mutants From left to right: lane 1, C139S; lane 2,
C176S; lane 3, C354S; lane 4, wild-type CFAS; lane 5, molecular mass
markers (from top to bottom, 66 kDa, 45 kDa, 36 kDa, 29 kDa,
24 kDa, 20.1 kDa).
Trang 6the enzyme in the presence of [methyl-3H]S-AdoMet but
without unsaturated phospholipids, for 3 h gave no more
counts in the water fraction than a control sample
containing no enzyme The detection limit of this
experi-ment was estimated at 0.2% exchange (i.e that an exchange
of 0.2% or more would have been easily detected) Addition
of cyclopropanated phospholipids in the reaction mixture
that could trigger a conformational change upon binding
did not enhance this exchange reaction The reverse
experiment, incorporation of solvent protons into the
substrate, which should be faster than the wash-out as no
intramolecular kinetic isotope effect on the abstraction
should occur, was tested using the same conditions but in
the presence of unlabeled S-AdoMet and deuteriated buffer
The reaction was followed by1H and13C-NMR, and again
no incorporation of deuterium could be detected Therefore,
under our conditions, CFAS is unable to catalyze the
exchange of the methyl protons of S-AdoMet, a result that
does not support the ylide mechanism
Thiol titration by DTNB
The amino acid sequence of wild-type E coli CFAS
predicts eight cysteines [9] The total thiol content of the
purified enzyme was spectrophotometrically titrated using
DTNB, in denaturing conditions using standard protocols
[29] A ratio of 7.5 ± 0.3 mol of free thiols per mol of
CFAS monomer was found, consistent with eight free
cysteines in the E coli wild-type CFAS In native
condi-tions (Fig 3), six thiols per monomer were titrated in one
hour with triphasic kinetics Three cysteines reacted within
4 min, two more cysteines reacted more slowly within
40 min, and one cysteine reacted in a third very slow phase
If the enzyme was left longer under these conditions (for
two further hours), the absorbance at 412 nm finally
reached a value compatible with eight free cysteines Therefore E coli CFAS contains three classes of free cysteines, three fast reacting thiols (exposed), two slowly reacting thiols (less accessible) and three buried cysteines that react extremely slowly The upward curvature of the trace in Fig 3, after 40 min (a reproducible phenomenon),
is probably due to a partial unfolding of the protein, exposing the buried cysteines, which consequently react faster It is not clear if the protein unfolds because of multiple chemical modifications or if it is simply due to the long incubation time
CFAS inactivation by DTNB
As already reported by Cronan and coworkers [9], we found that CFAS could be inactivated by thiol-directed reagents such as DTNB and N-ethylmaleimide (NEM) Kinetic analysis of the inactivation process by DTNB is shown in Fig 4 The inactivation follows a pseudo first-order kinetics with no saturation and with a second-order rate constant of 1.2M )1Æs)1, a low but not unprecedented value [32] Similar analysis using NEM showed that the inactivation occurred similarly with a rate constant of 2.4M )1Æs)1(not shown) The inactivation process was not significantly slowed down
in the presence of a saturating concentration of S-AdoMet (0.38 mM) or in the presence of 1 mgÆmL)1 unsaturated phospholipids (Table 1) This suggests that the cysteine residue responsible for the inactivation is not located in the active site
For the graphical analysis of the inactivation, His6-tagged wild-type CFAS (2.4 lM) was treated with excess DTNB (0.31 mM) at pH 7.3 and the residual activity together with the number of modified sulfhydryls per CFAS were determined at the same time points The data were analyzed graphically as described by Tsou (Fig 5) using the following Eqn (2) [30]:
(a)1=i¼ ðp þ s mÞ=p ð2Þ where m is the number of modified cysteines per monomeric CFAS, s the number of fast reacting cysteines that are nonessential, a the fraction of remaining activity when m residues have been modified, p is the number of cysteines reacting slower than the s group, and i is the number of essential residues for catalytic activity, as defined by Tsou Note that the i class belongs to the p class The graph shown
in Fig 5 confirms the presence of three classes of free cysteines in the enzyme First, three cysteines react quickly, with no loss of activiy, then two more cysteines react with concomitant loss of enzyme activity, and finally the buried cysteines react The portion of the graph where the activity
is lost perfectly fits to a straight line when i¼ 1 (the correlation coefficient is 0.99) When the same data are plotted with i¼ 2 or i ¼ 3 the data points clearly deviate from linearity Therefore the data of Fig 5 are most consistent with one cysteine, chemical modification of which leads to inactivation Futhermore the plot allows the estimation of s and p, as the ordinate intercept is (p+s)/
p¼ 2.1, the abscissa intercept is p+s ¼ 5 and the slope is )1/p ¼ –0.44 Thus p ¼ 2.3 and s ¼ 2.7, values in close agreement with the numbers deduced from Fig 3 (where
p¼ 2 and s ¼ 3)
0
1
2
3
4
5
6
7
Number of reacting thiol per monomer (mol/mol)
Time (min)
recorded against time.
Trang 7Characterization of C139S, C176S and C354S mutant
proteins
Alignment of the sequence of all cyclopropane
syntha-ses known so far shows that among the eight cysteine
residues of the E coli CFAS only three are strictly
conserved: C139, C176 and C354 [1] We thus prepared
three corresponding Cysfi Ser mutants for analysis This particular replacement was chosen because it is isosteric, but the OH group is much less acidic and much less nucleophilic
0.00
0.05
0.10
0.15
-1 )
[DTNB] (mM)
1 0
100
Time (min)
EDTA Aliquots were withdrawn at different time points and the
residual activity was measured (see Experimantal procedures for
details) Each data point represents the average of two independant
experiments, with less than 5% deviation from the mean Error bars
were calculated by fitting the data points to simple exponential decays.
inacti-vation in the absence of DTNB, were plotted against DTNB
concen-tration Data were fitted to a straight line.
Table 1 Summary of the inactivation rate constant in the presence of DTNB All experiments were run under the following conditions:
wild-type CFAS
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Number of titrated thiol per monomer
were monitored at the same time (see Experimental procedures) The
fitted to a straight line Each data point represents the average of two independent experiments Error bars are not shown for clarity See text for details of the analysis.
CFAS.
Enzyme
S-AdoMet
Relative catalytic efficiency (%)
Trang 8than the thiol group All mutant genes were obtained by
PCR amplification using two sets of mutated primers, and
the Stratagene technology The desired mutations were
verified by DNA sequencing and the mutated proteins were
expressed and purified as described for the His6-tagged
wild-type enzyme (Fig 1) Table 2 summarizes the kinetic
parameters for individual mutants and His6-tagged
wild-type enzyme It is clear that all mutant are active, the slowest
being C139S (16% relative catalytic efficiency) Therefore
none of the conserved cysteines is essential for the activity
The three mutants were inactivated by DTNB at a rate
similar to that measured for the His6-tagged wild-type
enzyme under the same conditions (Table 1) Thus, the
cysteine residue that is responsible for the inactivation is not
one of the conserved cysteines, a result compatible with the
fact that no protection was observed by the substrates
Discussion
The cyclopropyl group is present in number of natural
products and its unusual properties have always stimulated
chemists and biochemists [33,34] Biosynthesis of this
structural element follows diverse schemes, but the direct
methylenation of double bonds, catalyzed by cyclopropane
synthases, is one of the most interesting Most intriguing is
the chemical mechanism by which this class of closely
related enzymes effects the cyclopropanation The
carboca-tion mechanism, first described by Lederer [20], is
chemic-ally sound and can also be applied to other methyl
transferases found in mycobacteria that are homologous
with cyclopropane synthases, and which catalyze
modifica-tions of unsaturated lipids, such as the formation of
a-methylketo- or a-methylhydoxy- fatty acids [35,36]
However, it quickly appeared that addition of a sulfur
ylide, derived from S-AdoMet, to the double bond of the
fatty acid could be another plausible alternate reaction
mechanism [14,17,18,21] The two mechanisms differ from
one another not only in the order of making and breaking
bonds, but also in the type of intermediate formed Progress
has recently been achieved with cloning and purification
of the E coli enzyme [9], as well as solving the three
dimensional structure of M tuberculosis enzymes [22], and
reports of some mechanistic experiments [23,24]
We report here the purification and characterization of a
recombinant CFAS bearing an N-terminal His6-tag The
use of nickel affinity chromatography allowed rapid
preparation of pure enzyme in substantial amounts A
similar successful strategy was recently followed by Liu and
coworkers [24]
As the ylide mechansim would be most likely to involve
carbenoid transfer to a metal [21], we searched for metals in
the enzyme No cofactors, organic or metallic, were found, in
accordance with structural data obtained for the M
tuber-culosisenzymes (for which no in vitro catalytic activity has
ever been reported) The reaction mechanism must therefore
rely solely on side chain functional groups, and thus only
acid-base or nucleophilic catalysis must operate
The pH profile of the activity, in saturating conditions,
revealed two ionisable groups important for catalysis: a first
pKa1of 6.8 and a second pKa2of 8.7 Interpretation of pH
effects are most difficult, but it is interesting to note that a
carbonate (pK ¼ 6.4), bound in the active site of the
M tuberculosisenzymes, has been suggested to be the base necessary to abstract the methyl proton [22] It is then tempting to attribute the pKa1¼ 6.8, detected by kinetic means to the carbonate, that was proposed to be the base on structural grounds Alternatively, this pKacould be attrib-uted to a His residue, such as His266 (His167 in the
M tuberculosissequence), which lies in the active site and could participate in a proton relay Futher mutagenetic experiments are in progress to clarify this point
One of the best ways to prove the ylide mechanism would
be to show that the enzyme is able to catalyze the exchange
of the methyl protons of S-AdoMet in the absence of other substrates Numbers of enzymatic reaction mechanisms have been supported on this type of experimental grounds [37] However, under our conditions we did not observe such an exchange, even in the presence of the cycloprop-anated product that could mimic the second substrate and hence trigger a conformational change, a strategy that was successful in the citrate synthase case [37] Therefore, our data do not support the ylide mechanism Of course, one cannot exclude the possibility that the abstraction is promoted by a monoprotic base that exchanges its proton with the solvent very slowly However, Buist and coworkers reported feeding experiments, using deuteriated methionine and L plantarum cells, which were then interpreted by invoking an exchange of the methyl protons on the carbocation intermediate (Scheme 2A) but not on an ylide species [17,18] Such a fast exchange (33% exchange) would probably require a polyprotic base and a reversible forma-tion of the cyclopropane ring However, these experiments were conducted using whole cells and were dependent on growth conditions, and thus need to be confirmed on the isolated enzyme
We have also addressed the role, in catalysis, of the cysteines of the E coli enzyme It has been suggested in the literature that a cysteine could be important for catalysis [9] Indeed, a thiolate could either abstract a proton on the methyl group or stabilize the carbocation, or even form a covalent adduct (the base or the nucleophile in Scheme 2) The alignment of the sequence of all cyclopropane synthases known so far [1], shows that only three cysteines among the eight cysteine residues of the E coli enzyme are conserved: C139, C176 and C354 In the three dimensional structure of the homologous M tuberculosis enzymes [22], C35 which corresponds to C139 of E coli CFAS shares a hydrogen bond, by its N-H, to a carbonate in the active site C269 of the M tuberculosis enzyme, which corresponds to C354 in the E coli enzyme, was found to be located near the active site The third conserved cysteine, C72 in the M tuberculosis enzyme, corresponding to C176 in the E coli enzyme, is located near the S-AdoMet binding site but far from the active site Therefore, it was reasonable to postulate an important role in catalysis for these conserved residues Cronan and coworkers reported very briefly in a review [1] that mutation of Cys176 and Cys354 to Ala, in the E coli enzyme, gave active mutants No experimental data were reported and the third conserved cysteine, Cys139, was not mutated We thus decided to re-examine this issue in detail Our results show that, in the E coli enzyme, all of the eight cysteines are free and that they can be classified into three classes: three fast reacting cysteines, two cysteines reacting at
a moderate rate, and three cysteines reacting very slowly
Trang 9Futhermore, using the graphical analysis developed by Tsou
[30], we show here that only one cysteine of the two
moderately reacting residues, is responsible for the
inactiva-tion of CFAS by DTNB Thus, five cysteines react with
thiol-directed reagent in 40 min under our conditions, but
only one modification leads to inactivation The three
mutated enzymes, C139S, C176S and C354S, were prepared
by site-directed mutagenesis, and were found to be active, the
slowest (16% active) being C139S, primarily affected on its
catalytic constant If any of these cysteines were involved in
base catalysis or nucleophilic catalysis, the corresponding
serine mutant should have been at least a hundred to a
thousand times less efficient than the wild-type enzyme A
dramatic drop in activity is usually observed in CysfiSer
mutants of enzymes known to use the thiolate group as a
base (racemases) or as a nucleophile (methyl transferases)
[38–40] The fact that inactivation by DTNB of the His6
-tagged wild-type enzyme was not protected by substrates,
and that the three CysfiSer mutants prepared in this report
are inactivated by DTNB at the same rate as the His6-tagged
wild-type enzyme, shows that the cysteine responsible for the
inactivation cannot be C139, C176 or C354 There are five
other cysteine residues in the E coli enzyme, and it is not
possible at the moment to attribute the residue that is
responsible for the inactivation Furthermore, this chemical
inactivation does not seem to be related to catalysis
In conclusion, the findings reported here do not support
the ylide mechanistic proposal but further support the
carbocation mechanism Furthermore, it is shown here that
the conserved cysteines of E coli CFAS are not directly
involved in catalysis and that the inactivation observed after
chemical modification of another cysteine probably comes
from steric hindrance, that is not relevent to catalysis The
base and the nucleophile supposedly involved in the reaction
mechanism are very likely other residues or functional
groups, e.g E239 or the active site carbonate Futher
mutagenesis experiments are underway to explore these
hypotheses
Acknowledgements
We wish to thank Thierry Drujon and Diane Delaroche for technical
assistance in preparing E coli phopholipids and in constructing
mutants of E coli cyclopropane fatty acid synthase We are grateful
to Sabine Cornet for initial work on cloning the cfa gene We are
indebted to Dr Kenneth A Cornell and Dr Michael Riscoe for the
generous gift of plasmid pEXH6MTAN and for advice in preparing
S-AdoHcy nucleosidase This work was supported in part by the ACI
program of the Ministe`re de lEducation Nationale de la Recherche et
de la Technologie’, grant number 0693.
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