The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates fo
Trang 1thioesterase from Alcaligenes faecalis
Puja Shahi*†, Ish Kumar*‡, Ritu Sharma, Shefali Sanger and Ravinder S Jolly
Institute of Microbial Technology, Chandigarh, India
Long-chain acyl-CoA thioesterases (EC 3.1.2.2)
hydro-lyze acyl-CoA esters to nonesterified fatty acids and
coenzyme A (CoASH) [1] These are ubiquitously
expressed in bacteria, yeast, plants and mammals, and
in most cell compartments, such as endoplasmic
reticu-lum, cytosol, mitochondria and peroxisomes Several
unrelated thioesterases have been purified to
homogen-eity from plants, animals, and bacteria, and the
cDNAs encoding several of them have been cloned
and sequenced [2–7] Although the physiological func-tions of these enzymes remain largely unknown, it is speculated that they regulate lipid metabolism by maintaining appropriate concentrations of acyl-CoA, CoASH, and nonesterified fatty acids The only estab-lished function for acyl-CoA thioesterases is in the termination of fatty acid synthesis in eukaryotes [8] Two thioesterases, I and II, that cleave acyl-CoA molecules in vitro have been characterized from
Keywords
Alcaligenes faecalis; immunogold electron
microscopy; long-chain acyl-CoA;
p-nitrophenyl esters; thioesterase
Correspondence
R S Jolly, Institute of Microbial
Technology, Sector 39, Chandigarh 160 036,
India
Fax: +91 172 269 0585
Tel: +91 172 269 0908
E-mail: jolly@imtech.res.in
*These authors contributed equally to this
paper
†Present address
Department of Physiology and Biophysics,
University of Iowa, Iowa city, IA 52242,
USA
‡Present address
Department of Chemistry, Wesleyan
Univer-sity, Middletown, CT 06459, USA
(Received 19 January 2006, revised 12
March 2006, accepted 22 March 2006)
doi:10.1111/j.1742-4658.2006.05244.x
A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations In solution, the protein exists as homo-meric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa sub-units MS⁄ MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling It hydrolyzed satur-ated and unsatursatur-ated fatty acyl-CoAs of C12to C18chain length with Vmax and Kmof 3.58–9.73 lmolÆmin)1Æ(mg protein))1 and 2.66–4.11 lm, respect-ively A catalytically important histidine residue is implicated in the active site of the enzyme The thioesterase was active and stable over a wide range of temperature and pH Maximum activity was observed at 65C and pH 10.5, and varied between 60% and 80% at temperatures of 25–70C and pH 6.5–10 The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thio-esterase than p-nitrophenyl esters When assayed at 37 and 20C, the affin-ity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower tem-perature, but remained largely unaltered for palmitoyl-CoA
Abbreviations
DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); TEM, transmission electron microscopy.
Trang 2Escherichia coli Thioesterase I, encoded by the tesA
gene, is a periplasmic protein of 20.5 kDa and has an
active site similar to serine proteases, consistent with
its inhibition with di-isopropyl fluorophosphate [2,9]
Thioesterase II, encoded by the tesB gene, is a
tetra-meric protein with identical subunits of 32 kDa and is
insensitive to inhibition with di-isopropyl
fluorophos-phate
A histidine residue present at position 58 in
thioest-erase II has been implicated in the cleavage of the
thioester bond [10,11] Two thioesterases with striking
similarities in their physical properties to thioesterase I
and II have been reported from photosynthetic
bac-teria, Rhodopseudomonas sphaeroides [12,13]
Cho and Cronan [2] prepared null mutants of both
tesA and tesB in an attempt to determine the role of
these thioesterases The deletion of tesA, tesB or both
genes has no effect on the growth or lipid composition
of E coli However, the possibility of another enzyme
taking over the function of both enzymes or the
pres-ence of a third thioesterase in E coli has not been
ruled out The overexpression of either tesA or tesB to
levels that greatly exceed normal also has no effect on
the growth of E coli [2,11] It has also been shown
that E coli thioesterases are unable to cleave
acyl-ACPs in vivo [14]
Recently, Schulz and coworkers [15] provided some
evidence for the function of cytoplasmic thioesterase of
E coli in b-oxidation They showed that oleate is
mostly degraded via the classical, isomerase-dependent
pathway in E coli, but that a small amount of
2-trans-5-cis-tetradecadienoyl-CoA is diverted from the
path-way by conversion into 3,5-cis-tetradecadienoyl-CoA
by D3,D2-enoyl-CoA isomerase The 3,5- intermediate,
which would strongly inhibit b-oxidation if allowed to
accumulate, is hydrolyzed and the resultant
3,5-tetra-decadienoate is excreted into the growth medium In
another study, Zheng et al [16] coexpressed
thioest-erase II with (R)-3-hydroxydecanoyl–acyl carrier
pro-tein-CoA transacylase (PhaG, encoded by the phaG
gene) to clarify the physiological role of thioesterase II
3-Hydroxydecanoic acid was produced in E coli by
mobilizing PhaG By using an isogenic tesB (encoding
thioesterase II)-negative knockout E coli strain, CH01,
it was found that the expression of tesB and phaG can
up-regulate each other In addition, 3-hydroxydecanoic
acid was synthesized from glucose or fructose by
recombinant E coli harboring phaG and tesB This
study supports the hypothesis that the physiological
role of thioesterase II in E coli is to prevent the
abnormal accumulation of intracellular acyl-CoA
We have isolated a thioesterase from Alcaligenes
faecalis ISH108 and demonstrated its application in
chemoselective and racemization free deacylation of thiol esters [17] A faecalis was isolated from soil sam-ples during routine screening of micro-organisms for various biotransformation applications In this paper,
we describe the intracellular localization and character-ization of the thioesterase The wild-type expression of protein was sufficiently large to obtain milligram quan-tities of the protein from about 20 g of cells, which required only 1.5-fold, single-step purification to obtain a near homogeneous preparation
Results
Isolation and purification of thioesterase The thioesterase extracted from the cells with 1 m NaCl, as described in Experimental procedures, exhib-ited a specific activity of 4.72 lmolÆmin)1Æ(mg pro-tein))1 The enzyme could also be extracted by suspending freshly grown cells in 50 mm Tris⁄ HCl buffer saturated with butanol at pH 7.4 [specific activ-ity 2.5 lmolÆmin)1Æ(mg protein))1] Other extraction systems, used to extract thioesterase from the cells, included 1 m NaCl with 0.1% Triton X-100 [specific activity 2.82 lmolÆmin)1Æ(mg protein))1] and butan-1-ol-saturated Tris⁄ HCl buffer containing 0.1% Triton X-100 [specific activity 2.43 lmolÆmin)1Æ(mg pro-tein))1]
As the specific activity in 1 m NaCl extract was the highest, it was selected as the method of choice for the isolation of the enzyme SDS⁄ PAGE of the NaCl extract, run under reducing conditions, showed a prom-inent band (> 90%) at 22 kDa, which suggested that further purification of the protein could be achieved by size exclusion chromatography Preliminary investiga-tion using Sephadex G-75 (fractionation range 3–80 kDa) revealed that the native size of the protein was much larger as it moved into the void volume of the column This allowed ultrafiltration with a 50-kDa Centricon membrane (Amicon, Bedford, MA, USA) for concentration of the samples or buffer change In the first attempt, Sephacryl S-300 (fractionation range 10–1500 kDa) was selected for the purification of protein The NaCl extract obtained was desalted and concentrated using 50-kDa Centricon membranes The concentrated sample was loaded on the column pre-equilibrated with 50 mm Tris⁄ HCl buffer (pH 7.6) containing 150 mm NaCl The column was eluted with the same buffer at a flow rate of 24 mLÆh)1 Thioest-erase activity moved near the void volume of the col-umn (Fig 1A) Finally, the protein was purified on a Sepharose CL-4B (fractionation range 60–20 000 kDa) column (Fig 1B) SDS⁄ PAGE of the purified protein,
Trang 3run under reducing conditions, showed a single band at
22 kDa (Fig 2A) Sepharose CL-4B chromatography
resulted in 1.5-fold purification with 50% yield
Molecular mass of thioesterase The elution profile of the protein on gel filtration col-umns indicated the protein to be a large homomeric aggregate of 22-kDa subunits On the Sepharose CL-4B column, the thioesterase was eluted just after the higher-molecular-mass (2000 kDa) fraction of dextran blue and much before the standard protein, thyroglobulin (669 kDa) (Fig 1C) The inability of the protein to move
in native PAGE (anodic disc-PAGE using Tris⁄ glycine,
as electrophoresis buffer, pH 8.3, or cathodic disc PAGE using alanine⁄ acetic acid buffer, pH 4.5), run under non-reducing conditions, is also consistent with this observa-tion Finally, the aggregated structure of native protein was established by transmission electron microscopy (TEM) The purified sample was concentrated by repeated ultrafiltration using a Centricon 50-kDa mem-brane and suspended in water at a concentration of
600 lgÆmL)1 TEM was performed on a carbon grid using 2% aqueous uranyl acetate and 2% phosphotung-stic acid at pH 8.0 An electron micrograph showed granular structures with a mean diameter of 21.6 nm (Fig 3A) The size distribution is shown in Fig 3B
Intracellular localization of thioesterase
We carried out electron microscopic immunogold labe-ling studies with ultrathin sections of Alcaligenes cells
to localize thioesterase at the ultrastructural level Polyclonal antibodies, AbTE-N and AbTE-D, raised against purified native enzyme and the piece of gel cor-responding to the 22-kDa monomeric protein on SDS⁄ PAGE, respectively, were assayed for their specif-icity by western blotting AbTE-D antibodies were used to rule out any nonspecific binding that might have occurred with AbTE-N because of the aggregated nature of the native protein The purified enzyme was run on SDS⁄ PAGE, and, after electroblotting on to nitrocellulose membrane, it was probed with AbTE-N and AbTE-D (Fig 2C) Both antibodies identified the 22-kDa band corresponding to the monomer of thio-esterase enzyme on denaturing gel
Alcaligenes was grown to mid-exponential phase, and, after several dehydration steps, embedded in LR White resin, which was then dehydrated in several steps Optimal ultrastructural preservation required inclusion of 0.2% glutaraldehyde in the fixative; the reactivity of the antibody was not affected by glutaral-dehyde fixation Thin sections cut using an ultramicro-tome were incubated with primary antibodies followed
by nanogold labeled secondary antibody as described
in Experimental procedures and visualized under the transmission electron microscope
Fraction No
A
B
C
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0 100 200 300 400 500 600 700 800
Fraction No
0.0
0.1
0.2
0.3
0.4
0 100 200 300 400 500 600
Fraction No
0.0
0.1
0.2
0.3
0.4
0.5
0.6
a
Fig 1 Elution profile of thioesterase on gel filtration
chromatogra-phy (A) 13 · 425 mm Sephacryl S-300 column; (B) 13 · 290 mm
Sepharose S-4B column The column was pre-equilibrated with
100 m M Tris ⁄ HCl buffer, pH 7.6, containing 150 m M NaCl at a flow
rate of mLÆh)1 Then 250 lg protein was loaded and eluted in the
same buffer Fractions of 2.0 mL each were collected and assayed
for thioesterase activity as described in Experimental procedures (d)
activity units (C) 13 · 290 mm Sepharose S-4B column, flow rate
10 mLÆh)1 Peak A, Blue dextran; B, thioesterase; C, thyroglobulin.
Trang 4Different fields were observed, and the gold particles
were found to be exclusively present on the surface of
the cells The immunogold labeling was much more
abundant for cells where AbTE-N (Fig 4A,B) was used
as primary antibody than for those where AbTE-D
(Fig 4C) was used No labeling occurred in the control
cells where primary antibodies were derived from
pre-immunized serum (Fig 4D)
Does A faecalis have multiple thioesterases?
Total thioesterase activity could not be extracted from
the cells with NaCl even after several treatments To
find the presence of any other thioesterase, 10 g cells,
suspended in phosphate buffer, pH 7.0 (total volume
25 mL) were sonicated in three batches of equal volume
and fractionated into soluble and particulate fractions
by ultracentrifugation (100 000 g for 4 h) Most of the
thioesterase activity (> 80%) was present in the soluble
fraction, but the particulate fraction was also found to
be active SDS⁄ PAGE of both these fractions,
run under reducing conditions showed the presence of a
22-kDa protein (Fig 2B) The particulate fraction on
incubation with 1 mL phosphate buffer, pH 7.0,
con-taining 1 m NaCl, released most of the activity Total
proteins were combined and, after removal of
particu-lates, were concentrated to 2.5 mL by ultrafiltration
(3-kDa membrane; filtrate was devoid of thioesterase
activity) Then 1 mL of the concentrated protein was
applied to a Sephacryl S-300 gel-filtration column
(13· 530 mm), pre-equilibrated with Tris ⁄ HCl buffer,
pH 7.5, containing 150 mm NaCl at a flow rate of
24 mLÆh)1 and eluted in the same buffer Fractions of 1.0 mL volume were collected and assayed, but thioest-erase activity was detected only in the void volume SDS⁄ PAGE of the void volume under reducing condi-tions showed the presence of only 22-kDa protein Thus, we could identify only one thioesterase activity in
A faecalis, in contrast with E coli and Rhodopseudo-monas sphaeroides, each of which contained two thio-esterases; in addition, a third one has been implicated
in E coli [2]
Mass spectrometry Tandem MS was performed by Midwest Bio Services (Overland Park, KS, USA) on an LCQ Deca XP Plus ion trap mass spectrometer (ThermoFinnigan, Arcade,
NY, USA) SDS⁄ PAGE of purified protein was run under reducing conditions The thioesterase band at
22 kDa from the Coomassie-stained gel was excised and subjected to in-gel trypsinization The resulting peptide mixture was concentrated on a peptide trap col-umn and washed to remove salts and other impurities The peptides were separated on a microcapillary C18 reverse-phase chromatography column, and the eluted peptides sprayed directly into the mass spectrometer
MS⁄ MS spectral data were obtained and analyzed by comparing them with the NCBI nonredundant protein sequence database using turbosequest and peeks online software (http://www.bioinformaticssolutions com:8080/peaksonline/) The observed MS⁄ MS spectra did not match any peptide from E coli thioesterases or any other thioesterase in the database The following
14.2 20.1 24.0 29.0 36.0 45.0
45.0 36.0 29.0 24.0 20.1 14.2
kDa kDa 1 2
1 2 3
A
B
C
Fig 2 Purification of thioesterase,
fraction-ation of thioesterase activity and western
blot with antibodies to thioesterase (A)
Pro-tein samples run on 12.5% SDS ⁄ PAGE after
purification Lane 1, molecular mass marker;
lane 2, purified enzyme (B) Fractionation of
thioesterase The particulate and soluble
fractions, obtained by ultracentrifugation of
sonicated cells, were run on gel Lane 1,
purified thioesterase; lane 2, membrane
fraction; lane 3, soluble fraction (C)
West-ern blotting Lane 1, marker; lane 2,
antibod-ies raised against purified protein (AbTE-N);
lane 3, antibodies raised against gel purified
and denatured protein (AbTE-D) were used
as primary antibody followed by horseradish
peroxidase-conjugated anti-rabbit IgG.
Trang 5peptide fragments were obtained: YYDDNIWIAL
DYCDYYQLTHKPASLEK, LTKDAKYLEKAKET
EETGD + EQYLR, which showed identity with a
putative hydrolase from Bacteroides thetaiotaomicron
VPI-5482 (accession gi|29339943|gb|AAO77738.1|) [18]
Attempts at preparation of thioesterase null
mutants by chemical mutagenesis
An attempt was made to obtain thioesterase null mutant
by N-methyl-N¢-nitrosoguanidine mutagenesis using the
standard protocol [19], but without any success Approximately 10 000 colonies were screened in one set for any mutation in the gene encoding thioesterase enzyme with the help of plate assay [20] The mutants giving negative or ambiguous thioesterase assay results were further analyzed for thioesterase activity by the 5,5¢-dithio-bis-(2-nitrobenzoic acid) (DTNB) method
No mutant could be found that lacked thioesterase activity
Substrate specificity of thioesterase Thioesterase-catalyzed hydrolysis of acyl-CoA derivatives
The effect of chain length on the activity of thio-esterase was studied The initial rate of hydrolysis of a series of saturated acyl-CoA derivatives at different concentrations in the range 1.5–60 lm by thioesterase (0.2 lg) was determined At saturating concentrations, stearoyl-CoA (C18:0) was the most active substrate, with the rate of hydrolysis decreasing with decreasing chain length The acyl-CoAs of chain length longer than C18, were not studied The rates of hydrolysis of palmitoyl-CoA (C16:0) and myristoyl-CoA (C14:0) at saturating concentrations were similar
The enzyme showed very little activity towards octa-noyl-CoA, which required a higher concentration of enzyme for detectable activity No activity was observed with acyl-CoAs having chain length smaller than C8 The thioesterase also possessed activity towards unsaturated long-chain acyl-CoA derivatives
Vmax and Km values were determined by least-squares analysis of double-reciprocal plots of the data obtained from the corresponding Michaelis–Menten plots
Vmax and Km values were in the range 3.58–9.73 lmolÆmin)1Æ(mg protein))1 and 2.66–4.11 lm, respect-ively (Table 1)
Thioesterase-catalyzed hydrolysis of p-nitrophenyl
esters The assay mixture (1 mL) consisted of 400 lm p-nitro-phenyl derivative in 0.1 m phosphate buffer, pH 7.2, containing 0.1 m NaCl The reaction was started by the addition of 0.2 lg thioesterase Initial rates were determined by measuring the increase in A346 (e¼
4800 m)1Æcm)1), the isosbestic point of the p-nitrophe-nol⁄ p-nitrophenoxide couple, as described [21] The effect of the chain length of the p-nitrophenyl esters
on the activity of thioesterase was studied (Table 2) p-Nitrophenyl propanoate (C3:0) was found to be the most active substrate, with the activity decreasing sharply with increasing or decreasing chain length
Diameter(nm)
0
10
20
30
40
50
A
B
Fig 3 TEM The purified thioesterase was desalted and
concentra-ted by repeaconcentra-ted ultrafiltration using a Centricon 50-kDa membrane
and suspended in water at a concentration of 600 lgÆmL)1 TEM
was performed on the carbon grid using 2% aqueous uranyl
acet-ate and 2% phosphotungstic acid at pH 8.0 (A) Electron
micro-graph showing granular particles with mean diameter 21.6 nm (B)
Size distribution of the thioesterase particles in TEM Particle size
distribution was evaluated by measuring the diameter of 100
parti-cles The diameter was the mean of two right angled axes.
Trang 6p-Nitrophenyl acetate (C2:0) and p-nitrophenyl
hexano-ate (C6:0) were 30% as active as their (C3:0)
counter-part p-Nitrophenyl dodecanoate (C12:0) showed less
than 10% of the maximum activity, whereas
p-nitro-phenyl esters with chain length of more than C12were
not hydrolyzed by the thioesterase p-Nitrophenyl
pro-panoate was a threefold more active substrate than the
best acyl-CoA substrate, stearoyl-CoA It is interesting
to note that chain length specificity of Alcaligenes
thio-esterase for p-nitrophenyl esters was opposite to that
for acyl-CoA derivatives
The thioesterase activity and p-nitrophenyl esterase
activities appear to be co-resident for the following
reasons (a) Diethyl pyrocarbonate completely
inhib-ited both activities (b) p-Nitrophenyl propanoate provided protection against inhibition of thioesterase activity by diethyl pyrocarbonate to the extent of 74% The experiment was performed as follows To a pre-cooled solution of enzyme (100 lg) in 1 mL phosphate buffer, pH 6.0, was added 50 lm p-nitrophenyl prop-ionate, immediately followed by 1 mm diethyl pyrocar-bonate (from 1 m ethanol stock solution) A parallel experiment was run as a control in which the addition
of substrate was omitted The sample was incubated for 5 min, and a 5-lL aliquot of each sample was withdrawn and assayed for activity by the DTNB method using stearoyl-CoA as substrate (c) In an analogous manner, when stearoyl-CoA was used as
Fig 4 Transmission electron micrographs
of immunogold-labeled Alcaligenes treated
with various primary antibodies Alcaligenes
cells were embedded in LR white resin as
described in Experimental procedures Thin
sections were incubated with primary
anti-body raised against thioesterase, followed
by anti-rabbit IgG with conjugated nanogold
particles, and samples were analyzed under
the electron microscope Different fields
were viewed Arrowheads denote gold
parti-cles (A) Primary antibody AbTE-N raised
against purified native thioesterase (B)
Enlarged view of a single cell (bar, 200 nm).
(C) Primary antibody AbTE-D, raised against
a gel piece corresponding to the
thioest-erase band in SDS ⁄ PAGE (D) Control in
which preimmune serum was used as
pri-mary antibody.
Trang 7protecting agent and activity was assayed with
p-nitro-phenyl propanoate as substrate, 93% protection
against inhibition was observed
Long-chain acyl-CoAs are better substrates than
aryl esters
Stearoyl-CoA provided better protection against
inhi-bition of thioesterase activity by diethyl pyrocarbonate
than p-nitrophenyl propanoate, as shown above, which
indicated that the long-chain acyl-CoAs are better
sub-strates for the enzyme than aryl esters This was
fur-ther confirmed by substrate competition experiments
Aryl esterase activity against p-nitrophenyl propionate
(400 lm) was inhibited by 52% when it was measured
in the presence of 100 lm stearoyl-CoA, whereas the
presence of 200 lm p-nitrophenyl propionate in the
assay mixture of stearoyl-CoA (50 lm) had no effect
on the thioesterase activity, measured by the DTNB
method
Implication of a histidine residue in the active site of thioesterase
The effect of various protein-modifying agents on the activity of the thioesterase was studied (Table 3) Phe-nylmethanesulfonyl fluoride, a serine-active agent, had
no significant effect on the activity of the enzyme N-Bromosuccinimide caused complete loss of activity at
1 mm concentration, indicating the presence of catalyti-cally important residues such as tyrosine, tryptophan and histidine However, dimethyl (2-hydroxy-5-nitro-benzyl)sulfonium bromide, a tryptophan-modifying agent, and N-acetylimidazole, a tyrosine inhibitor, did not have any significant effect on the thioesterase activ-ity Diethyl pyrocarbonate, a histidine-modifying agent, caused total loss of activity at 1 mm concentration The thiol-reactive agent DTNB did not have a significant effect on the thioesterase activity, allowing the use of DTNB in the thioesterase assay Picrylsulfonic acid, a lysine-modifying agent, also had no effect on the activ-ity of the enzyme These results indicate the presence of
a catalytically important histidine residue in the enzyme The presence of a histidine residue was further supported by the following experiments
(a) Reversal of inhibition by hydroxylamine The activity of the enzyme could be partially recovered by the treatment of diethyl pyrocarbonate-inhibited enzyme with hydroxylamine An aliquot of enzyme, inactivated with 1 mm diethyl pyrocarbonate at 4 C, was incubated at 25C, for 8 h with 250 mm hydrox-ylamine and assayed for thioesterase activity by the DTNB method after extensive dialysis, as described in Experimental procedures Its activity was expressed as
a percentage of that obtained from an experiment, run in parallel, in which same amount of active enzyme (no inhibitor added) was incubated with
250 mm hydroxylamine for 8 h at 25C and assayed for activity in the same manner The treatment of the diethyl pyrocarbonate-inhibited enzyme with
Table 1 Thioesterase catalyzed hydrolysis of acyl-CoA derivatives.
A solution of acyl-CoA derivative was prepared in 100 m M Tris ⁄ HCl
buffer, pH 7.6, at various concentrations in the range 1.5–60 l M
The reaction was started by the addition of an aliquot of enzyme
(0.2 lg), and initial rates of hydrolysis were measured by the DTNB
method as described in Experimental procedures V max and K m
val-ues were determined by least-squares analysis of double-reciprocal
plots of the data obtained from the corresponding Michaelis–
Menten plots Sr No., serial number.
Sr No Substrate K m (l M )
V max [lmolÆmin)1Æ (mg protein))1]
5 Myristoleoyl-CoA 3.39 ± 0.3 4.77 ± 0.4
6 Palmitoleoyl-CoA 3.90 ± 0.1 5.57 ± 0.3
7 cis-Vaccenoyl-CoA 2.84 ± 0.3 6.05 ± 0.5
Table 2 Thioesterase-catalyzed hydrolysis of aromatic esters The assay mixture (1 mL) consisted of 400 l M p-nitrophenyl derivative in 0.1 M phosphate buffer, pH 7.2, containing 0.1 M NaCl The reaction was started by the addition of 0.2 lg thioesterase Initial rates were determined by measuring the increase in A346(e ¼ 4800 M )1Æcm)1), the isobestic point of the p-nitrophenol⁄ p-nitrophenoxide couple.
Specific activity [lmolÆmin)1Æ(mg protein))1]
Relative activity (%)
Trang 8hydroxylamine in this way resulted in 58.7% recovery
of activity
(b) A 35.7% increase in absorption at k245
corres-ponding to N-ethoxycarbonylation of histidine on
inactivation of thioesterase with diethyl pyrocarbonate
for 1 h was observed in differential spectra obtained as
described in Experimental procedures
(c) Stearoyl-CoA, a substrate of the enzyme provided
93% protection against inhibition with diethyl
pyro-carbonate, as shown above
Effect of temperature and pH on the activity of
the thioesterase
Optimum pH and pH stability
For determination of the optimum pH of the enzyme,
its activity was measured at various pHs ranging from
5.5 to 10.5 at 30C (Fig 5A) The maximum activity
of thioesterase was obtained at pH 10.5 For pH 5.5–
8.0, phosphate buffers were used, in which 80% of
maximum activity was retained For pH 7.2–9.0,
Tris⁄ HCl buffers were used Although the trend of
activity in the pH range 7.2–8.0 was the same as that
in phosphate buffer, the activity was 20% less than
that in phosphate buffer For pH 9.0–10.5, sodium
carbonate buffer was used At pH 9.0, the activity was
40% less in carbonate buffer than in Tris buffer
The effects of buffer composition on enzyme activity
have been reported previously [22] These effects may
be due to the effects of the buffer on the
oligomeriza-tion status of the enzyme [23] An alternative
explan-ation is that the binding affinity of the enzyme for the
substrate is modified, presumably because of differ-ences in the interaction of the buffer ions with the binding site [24] Activity at pH 10.5 was maximum and set as 100% Activity above pH 10.5 was not stud-ied Controls were used in each case to compensate for chemical hydrolysis, which was substantial at higher
pH Although maximum activity was obtained at
pH 10.5, all studies were carried out at pH 7.0–7.5 as the substrates are prone to degradation under basic conditions
To evaluate pH stability, the enzyme was incubated
in different buffers, pH 5.5–10.5, at 30C for 20 h The remaining activity is expressed as a percentage of the activity relative to the activity in the corresponding buffer at time zero Thioesterase retained almost
Table 3 Effect of protein-modifying reagents on thioesterase
activ-ity Purified and dialyzed thioesterase at a concentration of
20 lgÆmL)1was incubated with each reagent at 25 C for 15 min
and dialyzed against 50 m M Tris ⁄ HCl buffer, pH 7.5, at 4 C with
four buffer changes for 12 h Residual activity, percentage of the
original activity, was calculated by the DTNB method as described
in Experimental procedures.
Sr No Reagent (1 m M ) Residual activity (%)
2 Phenylmethanesulfonyl fluoride 90.7
6 Dimethyl (2-hydroxy-5-nitrobenzyl)
sulfonium bromide
89.0
9 5,5¢-Dithiobis-(2-nitrobenzoic acid) 98.4
pH
0 20 40 60 80 100 120
pH
20 40 60 80 100
120
A
B
Fig 5 (A) Effect of pH on the activity of thioesterase Assays were performed at 30 C in various buffers at different pH The activity
in carbonate buffer at pH 10.5 was set as 100%, all other values are relative to it (B) pH stability of thioesterase A predetermined amount of thioesterase was incubated in different buffers for 20 h
at 30 C and assayed for thioesterase activity The remaining activ-ity is expressed as percentage of activactiv-ity relative to the activactiv-ity in the corresponding buffer at time zero (d) phosphate buffer; (.) Tris⁄ HCl buffer; (n) carbonate buffer.
Trang 9complete activity at pH 5.5–6.0 (Fig 5B) It retained
80% activity at pH 7.0–7.5 and was relatively
unsta-ble under alkaline conditions After 20 h incubation at
pH 10.5 in carbonate buffer, only 20% activity was
retained
Thermal properties of the thioesterase
Thioesterase activity was determined at different
tem-peratures in phosphate buffer at pH 7.0 Maximum
thioesterase activity occurred at 65C (Fig 6A)
About 60–80% of maximum activity was retained at
temperatures of 25–70C There was sharp decline in
activity at 75–80C; the enzyme retained 20% of
maximum activity at 80C
To evaluate temperature stability, enzyme in
phos-phate buffer, pH 7.0 was incubated for 3 h at different
temperatures Thioesterase activity was assayed at
30C by the DTNB method as described in
Experi-mental procedures The activity at zero time at 30C
is assumed to be 100%, and all other values are
expressed relative to it Most of the enzyme activity
was retained at 25–50C (Fig 6B) After 3 h, 70% of
the activity remained at 70C At 80 C, most of the
activity was lost after 3 h
Effect of metal ions on thioesterase activity The effect of various bivalent metal ions on thioest-erase activity was studied by the DTNB method as described in Experimental procedures Zn2+ showed concentration-dependent partial inhibition of the enzyme It had no effect on the activity at 1 mm con-centration but caused 40% inhibition at 10 mm Hg2+ and Cu2+caused complete inhibition of enzyme activ-ity at 1 mm concentration The activactiv-ity was enhanced
by 10–20% when enzyme assays were performed in the presence of Mg2+, Ni2+or Co2+
Effect of temperature on the kinetics of thioesterase-catalyzed hydrolysis of palmitoyl-CoA, palmitoleoyl-CoA and cis-vaccenoyl-CoA
The cells were grown at 25, 30 and 37C and assayed for thioesterase activity by the DTNB method No difference in activity was observed, ruling out a tem-perature-dependent change in expression levels of thio-esterase The kinetics of thioesterase activity was determined at 20 and 37C with palmitoyl-CoA, palmitoleoyl-CoA or cis-vaccenoyl-CoA as substrate (Table 4) With palmitoyl-CoA as substrate, thioest-erase showed only marginal changes in Vmax and Km values at both the temperatures However, approxi-mately twofold reduced affinity and catalytic efficiency was observed when palmitoleoyl-CoA or cis-vaccenoyl-CoA was the substrate at the lower temperature
Discussion
Two thioesterases, I and II, that cleave acyl-CoA mol-ecules in vitro have been characterized from E coli Thioesterase I is a periplasmic protein of 20.5 kDa and has an active site similar to serine proteases [2,9] Thio-esterase II is a tetrameric protein with identical subunits of 32 kDa and is insensitive to inhibition with di-isopropyl fluorophosphate A histidine residue in thioesterase II has been implicated in the cleavage of the thioester bond [10,11] In comparison, thioesterase from A faecalis exists as large homomeric granular aggregates (21.6 nm average diameter) of 22-kDa sub-units (Figs 2A and 3) Phenylmethanesulfonyl fluoride,
a serine-active reagent, failed to inhibit the catalytic activity of thioesterase A faecalis thioesterase was digested with trypsin, and the resulting peptides separ-ated on a microcapillary C18 reverse-phase chromato-graphy column MS⁄ MS data were obtained and analyzed by comparing them with the NCBI non-redundant protein sequence database The observed
Temperature (oC)
0
20
40
60
80
100
120
A
B
Fig 6 Thermal properties of thioesterase (A) Optimum
tempera-ture of thioesterase activity Assays were performed in 50 m M
phosphate buffer, pH 7.0 The relative activity is expressed as
per-centage of maximum activity attained under the experimental
conditions (B) Thermostability of thioesterase A predetermined
amount of thioesterase was incubated for 3 h at different
tempera-tures and then assayed for thioesterase activity in 50 m M
phos-phate buffer at 30 C The activity at zero time at 30 C is assumed
to be 100%; all other values are expressed relative to it.
Trang 10MS⁄ MS spectra did not match any peptide from
E coli thioesterases or any other thioesterase in the
database Thioesterase was found to be associated
exclusively with the surface of cells as revealed by
ultrastructural studies using electron microscopic
im-munogold labeling studies (Fig 4)
A histidine residue has been implicated in the
active site of the enzyme based on the following
observations (a) Incubation of thioesterase with
1 mm diethyl pyrocarbonate resulted in complete loss
of enzyme activity (b) An increase in absorption at
k245 corresponding to N-ethoxycarbonylation of
histi-dine on inactivation with diethyl pyrocarbonate was
observed when differential spectra were recorded
(c) Approximately 60% of the enzyme activity could
be recovered by the treatment of diethyl
pyrocarbo-nate-inhibited enzyme with hydroxylamine (d)
Stea-royl-CoA, a substrate of the thioesterase, provided
93% protection against inactivation by diethyl
pyrocarbonate
Alcaligenesthioesterase was active in and stable to a
wide range of temperatures and pH values Maximum
activity was observed at 65C and pH 10.5 and varied
between 60% and 80% at 25–70C and pH 6.5–10
(Figs 5 and 6) Enzyme activity remained unaltered
after incubation in phosphate buffer for 3 h at 50C
Thioesterase hydrolyzed saturated and unsaturated
acyl-CoAs of C10 to C18 chain length with Vmax and
Km values in the range 3.58–9.73 lmolÆmin)1Æ(mg
pro-tein))1 and 2.66–4.11 lm, respectively (Table 1) At
saturating concentrations, stearoyl-CoA (C18:0) was the
most active substrate, with the rate of hydrolysis
decreasing with decreasing chain length Thioesterase
also has chymotrypsin-like activity and was able to
hydrolyze p-nitrophenyl esters of C2 to C12 chain
length The most active substrate was C3, with the
activity falling sharply with increase or decrease in
chain length Long-chain p-nitrophenyl esters were not
hydrolyzed by the enzyme The odd-chain C3esters are
unlikely to be natural substrates of the enzyme In any
case, the substrate competition experiments clearly
demonstrated that the long-chain acyl-CoAs are better substrates than p-nitrophenyl esters
The ratio of saturated⁄ unsaturated fatty acid in mem-brane phospholipids is tightly controlled in a tempera-ture-dependent manner in micro-organisms, which allows proper thermal regulation of membrane fluidity [25–27] Thermal regulation of membrane fluidity is common to all organisms Lower growth temperatures result in an increase in the number of unsaturated phospholipids in the membrane E coli can synthesize phospholipids almost entirely from exogenous fatty acids supplied by the growth medium The satur-ated⁄ unsaturated fatty acids in membrane phospho-lipids, synthesized from exogenous fatty acids is similar
to de novo ratio in a temperature-controlled fashion [28] A site for thermal regulation must therefore exist at the level of utilization of exogenous fatty acids, in addi-tion to a well-defined site for thermal regulaaddi-tion in
de novofatty acid synthesis [26] Previous literature sug-gests that such a regulation is likely to be at the enzyme and not gene level [29] Starting from exogenous fatty acids, the incorporation is known to involve first the formation of acyl-CoA derivatives Therefore, the possi-bility exists that a thioesterase may be involved in this thermal regulation, if it is able to control the ratios of saturated and unsaturated fatty acyl-CoAs in a tem-perature-dependent manner Vmax⁄ Kmvalues for palmi-toyl-CoA were 1.74 and 1.57 at 37 and 20C, respectively (Table 4) The corresponding values for palmitoleoyl-CoA were 1.43 and 0.64, and 2.13 and 0.92 for cis-vaccenoyl-CoA The Km values for palmitoyl-CoA at 37 and 20C were 4.11 and 3.85, respectively, whereas the corresponding values for palmitoleoyl-CoA were 3.90 and 7.20, and 2.84 and 5.51 for cis-vaccenoyl-CoA Whereas the affinity and catalytic efficiency of Al-caligenes thioesterase were reduced by about twofold for palmitoleoyl-CoA and cis-vaccenoyl-CoA at lower temperature, these remained largely unaltered for palmi-toyl-CoA, which should result in a higher ratio of unsat-urated⁄ saturated fatty acyl-CoA at lower temperature compared with higher temperature In principle, the
Table 4 Effect of temperature on the kinetics of thioesterase-catalyzed hydrolysis of palmitoyl-CoA, palmitoleoyl-CoA and cis-vaccenoyl-CoA A solution of acyl-CoA derivative was prepared in 100 m M Tris ⁄ HCl buffer, pH 7.6, at various concentrations in the range 1.5–60 l M The reaction was started by the addition of an aliquot of enzyme (0.2 lg), and initial rates of hydrolysis were measured by the DTNB method
as described in Experimental procedures Vmaxand Kmvalues were determined by least-squares analysis of double-reciprocal plots of the data obtained from the corresponding Michaelis–Menten plots.
Temp (C)