Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FIK

Article abstract of DOI:10.1074/jbc.M110.107177

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr⁴², His⁷⁶, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg¹²⁰ located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

Full text of DOI:10.1074/jbc.M110.107177

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 29, pp. 22495–22504, July 16, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Author’s Choice
Structural Basis for the Activity and Substrate Specificity of
Fluoroacetyl-CoA Thioesterase FlK
Received for publication, January 23, 2010, and in revised form, April 8, 2010 Published, JBC Papers in Press, April 29, 2010, DOI 10.1074/jbc.M110.107177
‡
1,2
§2,3,4
‡5
§6
‡7
§
Marcio V. B. Dias , Fanglu Huang
, Dimitri Y. Chirgadze , Manuela Tosin , Dieter Spiteller , Emily F. V. Dry ,
‡
‡
§†
Peter F. Leadlay , Jonathan B. Spencer , and Tom L. Blundell
‡
§
From the Department of Biochemistry and University Chemical Laboratory, University of Cambridge, Lensfield Road,
Cambridge CB2 1EW, United Kingdom
The thioesterase FlK from the fluoroacetate-producing Strep- S-adenosyl-L-methionine and fluoride (2–6). We have cloned
tomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coen- the fluorinase gene flA (5) and subsequently identified a gene
zyme A. This provides an effective self-defense mechanism, pre- cluster (fl) surrounding the fluorinase gene from S. cattleya (7).
venting any fluoroacetyl-coenzyme A formed from being Characterization of the 5Ј-fluoro-5Ј-deoxyadenosine phos-
further metabolized to 4-hydroxy-trans-aconitate, a lethal phorylase (3), the enzyme responsible for the second step of
inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does the FAc biosynthesis encoded by the flB gene located next to the
not accept acetyl-coenzyme A as a substrate. Crystal structure flA, has confirmed the involvement of the fl gene cluster in
analysis shows that FlK forms a dimer, in which each subunit the biosynthesis of FAc (7). In our efforts to further elucidate
adopts a hot dog fold as observed for type II thioesterases. the functions of the fl gene cluster, the protein encoded by the
Unlike other type II thioesterases, which invariably utilize either flK gene located close to the 3Ј-end of the cluster was charac-
an aspartate or a glutamate as catalytic base, we show by site- terized as a thioesterase that cleaves fluoroacetyl-coenzyme A
directed mutagenesis and crystallography that FlK employs a (FAcCoA) to produce FAc and CoA. It is noteworthy that
4
2
76
catalytic triad composed of Thr , His , and a water molecule,
analogous to the Ser/Cys-His-acid triad of type I thioesterases.
Structural comparison of FlK complexed with various substrate
analogues suggests that the interaction between the fluorine of
acetyl-coenzyme A (AcCoA) is not hydrolyzed by FlK (7). Pre-
vious studies have shown that FAc can be used by AcCoA syn-
thase to synthesize FAcCoA, and citrate synthase can catalyze
formation of 2-fluorocitrate from FAcCoA and oxaloacetate
1
20
the substrate and the side chain of Arg located opposite to the
catalytic triad is essential for correct coordination of the sub-
strate at the active site and therefore accounts for the substrate
specificity.
(8). Lauble et al. (9) have shown that the (Ϫ)-erythro diaste-
reomer of 2-fluorocitrate is further converted to 4-hydroxy-
trans-aconitate, a lethal competitive inhibitor of aconitase,
thereby blocking the tricarboxylic acid cycle. The presence of a
FAcCoA-hydrolyzing enzyme that has no effect on the meta-
bolically important intermediate AcCoA may therefore serve as
In 1986, Sanada et al. (1) discovered that Streptomyces cat- a self-resistance mechanism for the FAc-producing S. cattleya.
8
tleya is able to produce fluoroacetate (FAc) from fluoride.
FlK is homologous to many predicted thioesterases and
Attempts to elucidate the mechanism of this unusual transfor- hypothetical proteins according to protein-protein BLAST
mation have led to the discovery of a fluorinase enzyme cata- searches against a non-redundant protein sequence data base
lyzing the formation of 5Ј-fluoro-5Ј-deoxyadenosine from (7). However, none of these putative homologues has a defined
function. Two groups of thioesterases have been identified,
thioesterases I and thioesterases II, based on differences in their
Author’s Choice—Final version full access.
The atomic coordinates and structure factors (codes 3KX7, 3KX8, 3KV7, 3KV8, amino acid sequence, protein folds, and catalytic mechanisms.
3
KVZ, 3KW1, 3KUV, 3KUW, 3KVU, and3KVI)havebeendepositedintheProtein
Type I thioesterases have evolved within the ␣/␤-hydrolase fold
Data Bank, Research Collaboratory for StructuralBioinformatics, RutgersUni-
versity, New Brunswick, NJ (http://www.rcsb.org/).
This work is dedicated to the memory of Dr. J. B. Spencer, who passed away domain of multidomain proteins, such as type I fatty acid syn-
superfamily and are usually present as a product-releasing
†
1
on April 6, 2008.
thases, polyketide synthases, and nonribosomal peptide syn-
Recipient of a postdoctoral fellowship from the Conselho Nacional de Des-
envolvimento Cientifico e Tecnol o´ gico.
Both authors contributed equally to this work.
Supported by Biotechnology and Biological Sciences Research Council
Grant BBSB13357.
To whom correspondence should be addressed. Tel.: 44-1223-336710; Fax:
4-1223-336362; E-mail: fh223@cam.ac.uk.
Supported by Wellcome Trust Programme Grant 079281/Z/06/Z.
Recipient of the Herchel Smith Fund Fellowship.
Present address: Dept. of Bioorganic Chemistry, Max Planck Institute for
Chemical Ecology, Hans-Kn o¨ ll-Strasse 8, D-07745 Jena, Germany.
thases. Type I thioesterases use serine or cysteine as the cata-
2
3
lytic residue (10, 11). Type II thioesterases are discrete proteins
associated with activities of hydrolyzing short- or medium-
chain acyl-CoAs and exist either as a homodimer of a single hot
dog fold (12) or as a monomer comprising a duplicated hot dog
fold (13). It has been shown that the active sites of type II thio-
esterase enzymes contain either a glutamate or an aspartate
residue acting as a general base or nucleophile responsible for
4
4
5
6
7
8
The abbreviations used are: FAc, fluoroacetate; Ac, acetate; AcCoA, acetyl- the catalytic function (14, 15), although the precise catalytic
coenzymeA;FAcCoA, fluoroacetyl-coenzymeA;FAcCPan, fluoroacetylcar-
mechanism has remained controversial. FlK, a discrete protein
ba(dethia)-pantetheine; FAcOPan, fluoroacetyl oxa(dethia)-pantetheine;
CHES, 2-(cyclohexylamino)ethanesulfonic acid; WtFlK, wild type FlK;
catalyzing hydrolysis of a short-chain acyl-CoA (fluoroacetyl
SeMet, selenomethionine.
CoA), apparently belongs to the type II thioesterase family and
JULY 16, 2010•VOLUME 285•NUMBER 29
JOURNAL OF BIOLOGICAL CHEMISTRY 22495

Crystal Structure of Fluoroacetyl-CoA Thioesterase
is therefore expected to have a carboxylic side chain at the two primer pairs: T7-27/3Ј-5Ј primers for PCR-1 and T7T-27/
active site. Protein sequence alignment analysis has revealed 5Ј-3Ј primers for PCR-2. The T7–27 primer (5Ј-TAATAC-
8
42
50
61
that nine amino acid residues in FlK (Gly , Thr , Glu , Leu , GACTCACTATAGGGGAATTGT-3Ј) and T7T-27 primer
69
76
78
83
116
Gly , His , Ala , Gly , and Gly ) are all conserved in (5Ј-GCTAGTTATTGCTCAG CGGTGGCAGCA-3Ј) hybrid-
homologues that have a similar protein size to FlK (ranging ize, respectively, to the upstream or downstream regions of the
42
76
from 127 to 180 amino acids). The presence of Thr and His
multicloning site in pET28a(ϩ). Mutations were created in
50
together with Glu among the conserved residues led us to either the 3Ј-5Ј primer or the 5Ј-3Ј primer or both, depending
speculate that FlK may employ a variation of the classical cata- on the specific design for each individual mutant.
42
lytic triad-type mechanism with Thr (instead of a Ser or a
PCR was carried out using cloned Pfu DNA polymerase
76
50
76
Cys), His , and Glu (instead of an Asp). His would then (Stratagene) with 25 cycles of denaturation at 94 °C for 1
42
42
serve as a base to deprotonate Thr . The deprotonated Thr - min, annealing at 55 °C for 1 min, and extension at 72 °C for 1
is proposed to act as a nucleophile attacking the fluoro- min plus a final extension at 72 °C for 10 min. The resulting
O
␥1
acetyl carbonyl carbon of FAcCoA to initiate the hydrolysis PCR products were digested with appropriate restriction
50
reaction and the carboxylic side chain of Glu to neutralize the enzymes, purified by gel extraction (Qiagen), and inserted into
76
charge developed on the His side chain during the transition pET28a(ϩ) vector between the NdeI and BamHI sites. The
state of the reaction by hydrogen bonding with the imidazole inserts of the recombinant plasmids were verified by DNA
76
nitrogen of His (7). Although a hydroxyl derived from threo- sequencing. Constructs with the correct inserts were intro-
TM
nine rather than from a serine residue has been found to act as duced into E. coli Rosetta
(DE3)pLysS competent cells
the catalytic nucleophile in some proteinases, including the (Novagen) for protein expression.
individual activities of the proteosome (16), to the best of our
Expression and Purification of FlK and FlK Mutants—An
knowledge, no functionally characterized thioesterase has been overnight culture (10 ml) from a single colony harboring the
reported to use threonine in this way.
desired plasmid was used for inoculation of 1 liter of fresh LB
We have carried out site-directed mutagenesis targeting the medium containing kanamycin (50 ␮g/ml). The culture was
putative catalytic residues and defined structures by x-ray crys- incubated at 37 °C, 250 rpm until A ϭ 0.5–0.8 was reached.
6
00
tallography of the wild type and mutant FlK proteins both with Overexpression of the proteins was induced by 0.2–0.3 mM
and without bound analogues of FAcCoA. Our studies provide isopropyl 1-thio-␤-D-galactopyranoside overnight at 16 °C with
insights into the catalytic mechanism of FlK and the high sub- shaking at 220 rpm before being harvested by centrifugation.
strate specificity of FlK for FAcCoA over the closely related For incorporation of L-SeMet into FlK, 1 liter of fresh medium
AcCoA.
containing 6 g of Na HPO , 3 g of KH PO , 1 g of NH Cl, 1 g of
2
4
2
4
4
NH Cl, 0.5 g of NaCl, 1 mM MgSO , 4 g of glucose, 0.5 mg of
4
4
EXPERIMENTAL PROCEDURES
thiamine vitamin B1, and 50 mg of kanamycin was inoculated
Cells, Reagents, and Equipment—All oligonucleotide primers with cells from a 10-ml overnight culture and incubated at 37 °C
were synthesized by MWG-Biotech. Restriction enzymes were with shaking at 250 rpm. When the cell density reached A
ϭ
00
6
purchased from New England Biolabs, T4 DNA ligase was from 0.3–0.5, a mixture of L-lysine (100 mg), L-phenylalanine (100
Fermentas Life Sciences, and cloned Pfu DNA polymerases mg), L-threonine (100 mg), L-leucine (50 mg), L-isoleucine (50
were from Stratagene. The following reagents were purchased mg), L-valine (50 mg), and L-SeMet (50 mg) was added into the
from Sigma: AcCoA, 5,5Ј-dithio-bis(2-nitrobenzoic acid) (Ell- culture, and incubation was continued for 30 min at 37 °C. Iso-
man’s reagent), CHES, ethylene glycol, polyethylene glycol propyl 1-thio-␤-D-galactopyranoside was then added to a final
4
000, polyethylene glycol monomethyl ether 2000, potassium concentration of 1 mM to induce protein overexpression over-
chloride (KCl), L-selenomethionine (L-SeMet), trisodium cit- night at 16 °C, 200 rpm. The cells were broken by sonication for
rate, Tris, sodium acetate, and sodium fluoroacetate. Amino 4 min (1 s on, 10 s off) to release overexpressed recombinant
acids used for cell cultures were from DUCHEFA Biochemie. protein. The supernatant of the cell lysate was applied to a His-
2
ϩ
FAcCoA was synthesized as described previously (7). An ÅKTA Bind௡ column (Novagen) charged with Co ions. Nonspecific
FPLC system and a HiLoad Superdex S200 column (16 ϫ 60 proteins were removed by washing the column with washing
cm) (GE Healthcare) were used for gel filtration purification of buffer (0.5 M NaCl, 40 mM imidazole, 20 mM Tris-HCl, pH 7.9),
proteins. The protein concentration was determined using the and the recombinant protein was eluted with elution buffer
Bradford method (Sigma). DNA sequencing was performed in (0.5 M NaCl, 200 mM imidazole, 20 mM Tris-HCl, pH 7.9). The
the Department of Biochemistry DNA Sequencing Facility buffer containing the purified proteins was exchanged with a
(
University of Cambridge) on an Applied Biosystems 3730xl buffer containing 100 mM KCl and 20 mM Tris-HCl, pH 7.5, and
DNA analyzer. Escherichia coli NovaBlue or DH10B cells were concentrated at 4 °C to a total volume of 1–2 ml. The His tag of
TM
used for cloning, and E. coli Rosetta
(DE3)pLysS cells were the proteins was removed by thrombin (restriction grade;
Novagen) digestion at room temperature overnight. The tag-
used for protein expression.
¨
Cloning of FlK and Mutants—The cloning of the recombi- free proteins were further purified by gel filtration on an AKTA
TM
TM
nant His-tagged wild-type FlK (WtFlK) in pET28a(ϩ) (Nova- Explorer FPLC system using a HiLoad
16/60 Superdex
gen) was described in our previous study (7). Mutants of FlK 200 Prep Grade column and a mobile phase containing 100 mM
each containing a single amino acid mutation were created by KCl and 20 mM Tris-HCl, pH 7.5. The purity of the proteins was
PCR using the WtFlK-expressing plasmid as a template. For checked by SDS-PAGE. The protein masses were confirmed by
each mutant, two PCR amplifications were carried out using liquid chromatography-electrospray ionization-mass spec-
2
2496 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 29•JULY 16, 2010

Crystal Structure of Fluoroacetyl-CoA Thioesterase
trometry on a 2.0 ϫ 250-mm Jupiter 5-␮m C4 column (Phe- Both mutants (T42SFlK and T42AFlK) and WtFlK were crys-
nomenex) using a Finnigan LCQ (Thermo Finnigan) coupled tallized under the same conditions.
with an HP1100 high pressure liquid chromatography system
Crystallization of T42SFlK in Complex with AcCoA—After
(
Agilent) with a flow rate of 0.2 ml/min and the following ϳ6 months, crystals of T42SFlK⅐AcCoA complex grew in con-
mobile phase gradient: 0–5 min, 35–45% B; 5–25 min, 45–75% ditions similar to those for the WtFlK⅐Ac complex except where
B; 25–30 min, 75–95% B (buffer A: 0.1% trifluoroacetic acid in the acetate was replaced with 20 mM AcCoA.
H O; buffer B: 0.1% trifluoroacetic acid in acetonitrile).
Crystallization of WtFlK in Complex with Analogues of
2
Enzymatic Assays—The thioesterase activity of FlK and its Fluoroacetyl Pantetheine—The protein was incubated with 5
mutants on FAcCoA was measured by monitoring spectropho- mM either FAcCPan or FAcOPan. Initial trials were carried out
TM
tometrically the increase in absorbance at 412 nm due to the using a Cartesian HONEYBEE
81 crystallization robot
reaction of released CoASH with 5,5Ј-dithio-bis(2-nitroben- (Genomic Solutions), and the protein was screened using the
zoic acid with a CARY 100 Bio UV-visible spectrophotometer crystallization conditions from SM1 (Nextal), Classics,
(
Varian) as described previously (7). All assays were performed pHclear, and pHclear II (Qiagen) crystallization solution kits. In
at 25 °C in a total volume of 0.5 ml in a spectrophotometer each reservoir of the 96-well CrystalQuick crystallization plate
cuvette with a 1-cm light path.
with square sitting drop position (Molecular Dimensions), 100
Preparation of Fluoroacetyl Carba(dethia)-pantetheine and ␮l of the crystallization solution was added. The drops con-
Fluoroacetyl Oxa(dethia)-pantetheine—To prepare fluoro- tained 100 nl of the reservoir solution and 100 nl of protein
acetyl carba(dethia)-pantetheine (FAcCPan), monofluorina- solution at 10 mg/ml in 50 mM Tris-HCl and 100 mM KCl, pH
tion of (O,OЈ-diacetyl)-malonyl carba(dethia)-pantetheine 7.5. The best hits without acetate appeared in a condition that
TM
methylester with Selecfluor
was carried out, followed by comprised 100 mM CHES, pH 9.5, and 1 M trisodium citrate.
base-catalyzed loss of the protecting groups and concomitant After optimization, the best crystals were obtained in a condi-
deprotection and decarboxylation of the malonyl moiety. tion containing 100 mM CHES, pH 9.5, and 0.8 M trisodium
Fluoroacetyl oxa(dethia)-pantetheine (FAcOPan) was obtained citrate with a protein concentration of 10 mg/ml in the pres-
from 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cou- ence of 10 mM FAcCPan or FAcOPan.
pling of O,OЈ-isopropylidene-oxa(dethia)-pantetheine with
Data Collection and Structure Determination—Data for apo-
sodium fluoroacetate, followed by acetal deprotection with form WtFlK and SeMet FlK were collected at beamline ID14.4,
Dowex 50-X8-400.
European Synchrotron Radiation Facility (Grenoble, France).
Crystallization of SeMet FlK in the Apo-form—The crystalli- The data sets were processed using DENZO and SCALEPACK
zation trials with the SeMet FlK protein at a concentration of 20 (version 1.97) (17). The data were truncated and converted to
mg/ml in 50 mM Tris-HCl and 100 mM KCl, pH 7.5, were car- structure factors using TRUNCATE (18) from the CCP4 suite
TM
ried out using a Cartesian HONEYBEE
81 crystallization (19). X-ray data for complexes of WtFlK⅐FAc, T42AFlK⅐FAc,
robot (Genomic Solutions), and protein crystallization was T42SFlK⅐AcCoA, WtFlK⅐FAcCPan, and WtFlK⅐FAcOPan were
screened using the crystallization conditions from SM1 (Nex- collected at the Swiss Light Source, and the WtFlK⅐Ac,
tal) and the Classics (Qiagen) crystallization solution kits. To T42SFlK⅐Ac, and T42SFlK⅐FAc were collected at the European
each reservoir of 96-well CrystalQuick crystallization plates Synchrotron Radiation Facility. The data sets were processed
with square sitting drop position (Molecular Dimensions) were using the program Mosflm (20) and scaled by SCALA (21). The
added 100 ␮l of the crystallization solution and 100 nl of protein structure of SeMet FlK in its apo-form was solved using single-
solution. The best crystals appeared after approximately 1 week wavelength anomalous diffraction and the Phenix program
in 0.1 M Tris, pH 7.5, and 20% polyethylene glycol monomethyl (22), and the other structures were solved by molecular
ether 2000.
replacement using the apo-structure as probe search in the
Crystallization of Wild-type FlK (WtFlK) and Its Mutants program AMoRe (23) in the CCP4 suite (19). The refinement
T42SFlK and T42AFlK) in Complex with Acetate (Ac) and FAc— was carried out using the program REFMAC 5.2 (24). Visual
(
Initially, crystallization trials were carried out using hanging inspection and water addition were performed using Xtal-
drop vapor diffusion at 293 K. The protein at 10 mg/ml in 50 View/xfit (25) and Coot (26). The quality of the model was
mM Tris-HCl buffer, pH 7.5, containing 100 mM KCl was assessed using PROCHECK (27). The figures were made
screened against mother liquor solutions from the Classics using PyMOL (28).
suite (Qiagen) and kept at 20 °C. The initial crystals were
RESULTS
obtained in 30% polyethylene glycol 4000, 0.1 M sodium acetate,
and 0.1 M Tris-HCl, pH 7.0. After optimization, the best crystals
Site-directed Mutagenesis—To investigate the reaction
were obtained with protein at 5 mg/ml in the same crystalliza- mechanism of FlK, mutants of FlK were created at residues
4
2
50
76
tion condition. The drops comprised 1 ␮l of protein solution Thr , Glu , and His , candidates for the catalytic triad in the
and 1 ␮l of crystallization solution, and the crystals appeared FlK protein. To assess the involvement of these residues in the
after 3 or 4 days. To crystallize the FlK in complex with FAc, the catalytic mechanism, the activities of the mutants were assayed
sodium acetate present in the crystallization solution was and compared with that of WtFlK. Four mutants of the FlK
replaced by 0.2 M sodium fluoroacetate. The crystals for both protein carrying single amino acid mutations (T42S, T42A,
complexes were flash-frozen in liquid nitrogen to perform the E50A, and H76D, respectively) were cloned in pET28a(ϩ) and
TM
data collection after soaking in cryoprotectant solution com- overexpressed in E. coli Rosetta
(DE3)pLysS cells as His-
2ϩ
posed of 25–30% ethylene glycol and 75–70% well solution. tagged proteins. All proteins were purified by Co -charged
JULY 16, 2010•VOLUME 285•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 22497

Crystal Structure of Fluoroacetyl-CoA Thioesterase
TABLE 1
Comparison of wild-type and mutant FlK
Enzyme reactions were carried out using FAcCoA as the substrate. Errors quoted represent the S.E. of curve fitting (Lineweaver-Burk plot). Each data point is the average
of four (WtFlK), three (T42SFlK), or two E50AFlK measurements.
FAcCoA concentration above which
Protein
Soluble protein
kcata
Km
kcat/K b
m
substrate inhibition was observed
mg/liter
sϪ1
␮M
mMϪ1 sϪ1
␮M
WtFlK
ϳ10–12
ϳ10–12
ϳ2
0.044 Ϯ 0.001
30 Ϯ 1.3
15 Ϯ 1.2
1.47 Ϯ 0.10
T42SFlK
T42AFlK
E50AFlK
0.409 Ϯ 0.159
27.3 Ϯ 12.8
12
19
ϳ2
0.111 Ϯ 0.041
206 Ϯ 67
0.54 Ϯ 0.37
a
b
The units are number of CoA released/s/enzyme molecule.
m
The error values given for kcat/K are calculated from the sum of the relative errors on values in the numerator and denominator.
His-Bind resin (Novagen) followed by gel filtration. Both the product analogue Ac. Although attempts to crystallize T42S
WtFlK and T42SFlK were expressed as soluble proteins with a and T42A mutants in their apo-forms failed, crystals of
yield of over 10 mg from 1 liter of culture. T42AFlK and T42SFlK in complex with Ac, FAc, or AcCoA and T42AFlK
E50AFlK mutants yielded only 2 mg of soluble enzyme/liter of with bound FAc were obtained, and their structures were
culture, although their expression levels were comparable with solved.
those of WtFlK and T42SFlK. During storage at 4 °C T42SFlK
Overall Fold and Oligomeric Association of FlK—The crystals
and T42AFlK also showed a much higher tendency to aggregate of FlK diffracted at resolutions between 2.35 and 1.5 Å and
than WtFlK. The recombinant H76DFlK protein expressed in belong to space group C2 or P2 with two, four, or eight FlK
1
E. coli was completely insoluble, so it was not used for further molecules in the asymmetric unit, depending on the nature
studies. All of the mutations seemed have disturbed protein of the complex. Those complexes that crystallized in C2
folding, with the H76D mutant being the most affected. The (WtFlK⅐Ac, WtFlK⅐FAc, T42SFlK⅐Ac, T42SFlK⅐FAc, and
molecular weights of the purified proteins were determined by T42AFlK⅐FAc) have a dimer in the asymmetric unit with a local
liquid chromatography-electrospray ionization-mass spec- 2-fold symmetry axis. The crystals in P2 have either dimers
1
trometry. Retention times on gel filtration through a Superdex (two in T42SFlK⅐AcCoA and apo-SeMet WtFlK) in the asym-
S200 column indicate that purified WtFlK and the T42AFlK, metric unit, also with local 2-fold symmetry, or two tetramers
T42SFlK, and E50AFlK mutants are all dimers.
(WtFlK⅐FAcCPan and WtFlK⅐FAcOPan) with orthogonal
The enzymatic characterization of WtFlK using FAcCoA as 2-fold axes giving 222 pseudosymmetry (Fig. 1, A and B). Oper-
substrate has been reported previously (7). The present study ation of the crystallographic 2-fold axis on the dimers in the
42
demonstrated that replacing the Thr42 with Ala in FlK abol- asymmetric unit in space group C2 generates a tetramer, iden-
ishes the enzyme activity, supporting our hypothesis that the tical to that observed in the WtFlK⅐FAcPan or WtFlK⅐FAcOPan
42
Thr -O may act as the catalytic nucleophile that attacks the crystal structures. The observation that these crystal structures
␥1
carbonyl thioester carbon of FAcCoA to initiate the hydrolysis contain discrete tetramers suggests that the protein may have
reaction. Not surprisingly, T42SFlK is still able to hydrolyze dimer-tetramer equilibrium in solution in which tetramers pre-
Ϫ1 Ϫ1
FAcCoA with a k /K value of 27.3 mM
s
, which is about dominate at high concentrations.
cat
m
Ϫ1 Ϫ1
1
9 times higher than that for WtFlK (1.47 mM
s
), and a
The protomer of FlK has a hot dog fold that comprises a
relatively smaller K value (15 ␮M, 50% lower than that for five-strand antiparallel ␤-sheet (the bun) wrapping around a
m
WtFlK (30 ␮M)). It is noteworthy that T42SFlK activity was central ␣-helix (the sausage) (Fig. 1A). The hot dog fold was first
inhibited at a substrate concentration above 12 ␮M. These observed for the structure of ␤-hydroxydecanoyl thiol ester
42
results suggest that the Ser -O␥ in T42SFlK is able to carry out dehydratase (12). A diverse range of enzymes with distinct cat-
4
2
the nucleophilic attack and that the absence of the Thr -C␥ in alytic activities has been found to have this fold. Thioesterases
the T42S mutant may have led to decreased ability to ensure are important members of the hot dog superfamily and include
correct substrate binding, resulting in the substrate inhibition. 4-hydroxybenzoyl-CoA thioesterases from Pseudomonas sp.
Ϫ1 Ϫ1
The k /K value of E50AFlK (0.54 mM
s
) is only 37% of CBS-3 (29) and from Arthrobacter sp. SU (30), Escherichia coli
cat
m
that for WtFlK, whereas the K (206 ␮M) is much higher than thioesterases II (13), and acyl-CoA thioesterases YciA from
m
that for WtFlK. E50AFlK also exhibited substrate inhibition Hemophilus influenzae (HI0827) (31). Despite the conserved
when the FAcCoA concentration was above 19 ␮M, suggesting hot dog fold, these thioesterases share a very low degree of
50
that Glu plays an important role in substrate recognition identity at amino acid sequence level. The FlK asymmetric unit
(
Table 1).
contains a dimer with pseudosymmetry about a 2-fold axis as
FlK Crystal Structure Determination—In order to under- observed previously in the structure of 4-hydroxybenzoyl-CoA
stand the catalytic mechanism of FlK, in particular how FlK thioesterase from Pseudomonas sp. (29). and (R)-specific enoyl-
distinguishes between FAcCoA and the structurally very simi- CoA hydratase from Aeromonas caviae (32). Most other thio-
lar AcCoA, 10 crystal structures of WtFlK, T42SFlK, and esterases with the hot dog fold form tetramers, although hex-
T42AFlK with or without various ligands were determined. The americ structures have also been observed (31, 33). The main
WtFlK structures were solved in the apo-form and in complex contacts between the two protomers in the FlK dimer are
with either the substrate analogue AcCoA or the substrate frag- through strand 2 (residues 70–76) of the two protomers. The
ment analogues FAcCPan and FAcOPan, the product FAc, or ␤-strands of the two protomers form together a 10-strand
2
2498 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of Fluoroacetyl-CoA Thioesterase
protomer, suggesting strong inter-
actions between them. These obser-
vations have led us to assume that
the minimal active unit of FlK is a
dimer.
The tetrameric structure ob-
served for WtFlK⅐FAcCPan is
formed by a dimer of dimers with
pseudo 222 symmetry (Fig. 1B).
The main interaction regions
are the 10-strand ␤-sheets present
in the dimers, which are posi-
tioned back to back. Non-polar
interactions predominate, but
additionally there are some water-
mediated hydrogen bonds. Similar
tetramers have been observed in
FIGURE 1. Overall structure of FlK depicted in a schematic representation. A, structure of WtFlK dimer. The
two protomers of the dimer are shown in green and orange, respectively. B, tetrameric structure (dimer of other thioesterases (13, 30, 34, 35).
dimers) of WtFlK in complex with the substrate analogue FAcCPan. The FAcCPan molecule is shown as sticks.
The contact area between two
The two protomers of the dimer on the top are indicated in green and orange, respectively. A red/blue color
scheme is used for the dimer at the bottom.
2
dimers is ϳ3300 Å , corresponding
to about 30% of the total solvent-ac-
cessible area of each dimer, further
suggesting the possibility of an equi-
librium between dimers and tet-
ramer in solution.
The Active Site—Consistent with
the results of the site-directed
mutagenesis of FlK described above,
the crystal structure of apo-form
WtFlK shows that side chains of the
4
2
candidate catalytic residues Thr
7
6
and His in one protomer and that
5
0
of Glu from the adjacent pro-
tomer cluster between the dimer
interface into the region between
the “bun” and the “sausage” (Fig. 2,
A and B). As expected, the O of
4
2
76
␥1
FIGURE 2. Catalytic center of FlK. A, the catalytic triad of FlK is composed of Thr , His (shown as sticks), and
4
2
76
a conserved water molecule (Wat) (shown as a sphere). Green and orange colors indicate the two protomers of Thr and the N of His are
␦
1
the dimer, respectively. B, the catalytic triad is located at the dimer interface. The electron density map used
was 2F Ϫ 2F .
within hydrogen bonding distance
2.74 Å). To our surprise, we
observed that the carboxylic group
o
c
(
5
0
42
␤
-sheet with the putative active site at the interface between the of Glu is located on the opposite side of Thr , where it cannot
7
3
76
protomers. A disulfide bond formed by their respective Cys
have any direct contact with His . However, a water molecule
1
residues covalently connects the two subunits. Wrapping (Wat ) is in a perfect position to form a hydrogen bond with the
72
74
76
around the disulfide bond are the Ile and the Val from both N of His (2.79 Å). This water molecule is conserved in the
␦
2
protomers with their side chains within hydrophobic interac- crystal structures of wild-type and mutant FlK with or with-
tion distance of each other. The C of the putative catalytic out bound ligands. Although this observation was unex-
␥2
42
72
Thr also makes a hydrophobic contact with Ile -C of the pected, it is in good agreement with our site-directed
␥2
partner protomer. All of these interactions contribute to bring- mutagenesis results. A water molecule replacing the acidic
ing the two protomers together. Furthermore, there are two salt residue in this type of catalytic triad was also found in the
21
bridges formed between the two protomers; the Lys -N inter- HAV-3C gene product, an ␣-chymotrypsin-like protease
␨
29
135
42
acts with the Glu -O , and Lys -N interacts with the car- produced by the hepatitis A picornavirus (36). The Thr -
⑀
2
␨
111
108
76
1
boxyl O groups of Asp and Asp in the partner protomer. His -Wat network is further stabilized by a hydrogen bond
␦
2
3
0
32
1
80
Hydrogen bonds formed by Ser -O and Glu -O with between Wat and Thr side chain hydroxyl and another
␥
⑀1
51
43
50
2
Trp -N , Gly -N with Glu -O through a water molecule, conserved water molecule (Wat ) that also forms a hydrogen
⑀
2
1
⑀2
7
44
40
76
1
80
2
and Tyr -OH with Phe -O also serve to stabilize the FlK bond with the Phe -N. Around the His -Wat -Thr /Wat
2
39
40
78
79
dimer. The contact area between two protomers is ϳ1750 Å , network, the side chains from Val , Phe , Ala , Ala , and
1
13
corresponding to 30% of the accessible surface area of each Ile
form a hydrophobic shield, protecting the network
JULY 16, 2010•VOLUME 285•NUMBER 29
JOURNAL OF BIOLOGICAL CHEMISTRY 22499

Crystal Structure of Fluoroacetyl-CoA Thioesterase
density maps of the crystals ob-
tained from co-crystallization of
WtFlK with FAc and CoA indicates
that only FAc was bound. Compari-
sons of the structure of WtFlK with-
out bound ligand with those of
WtFlK⅐FAcCPan, WtFlK⅐FAcOPan,
and T42SFlK⅐AcCoA have provided
interesting insights into the struc-
tural basis of substrate recognition
by FlK.
In the ligand-free WtFlK crystal
structure, three well conserved
FIGURE 3. Active sites of ligand-bound FlK with 2F ؊ 2F electron density map for T42SFlK⅐AcCoA (A),
WtFlK⅐FAcCPan (B), and WtFlK⅐FAcOPan (C). The molecules of AcCoA, FAcCPan, and FAcOPan are all shown
in a deep purple color. Green and orange colors indicate residues from the two protomers of the dimer.
o
c
3
water molecules (designated Wat ,
4
5
Wat , and Wat ) mediate a hydro-
4
2
gen bonding network linking Thr
50
4
3
and Gly in one protomer to Glu ,
1
20
69
Arg , and Gly in the neighboring
3
43
protomer; Wat links the Gly -N
5
0
4
with the Glu -O , and Wat and
⑀2
5
Wat are within hydrogen bonding
distance of each other and of the
4
2
69
Thr -N and Gly -N, respectively.
1
20
The Arg -NH joins the network
1
5
0
by salt bridging with Glu -O and
⑀1
5
hydrogen bonding with Wat . Con-
1
20
120
verting Arg
to Ala
yielded
completely insoluble protein, as did
5
0
50
converting Glu to Ala , suggest-
ing the importance of these interac-
tions in maintaining the correct
protein folding and/or the correct
architecture of the active site cavity.
In the T42SFlK⅐AcCoA complex,
the AcCoA is sandwiched between
the long ␤-sheet and residues
1
7–42 that form two small ␣-heli-
ces (Fig. 4B). However, only the
acetyl and ␤-mercaptoethylamine
moieties from acetyl-CoA are bur-
ied in the protein, whereas the pan-
tothenic acid and the 3Ј-phos-
phoryl-ADP are exposed to solvent,
allowing considerable flexibility
(Fig. 4, A and B). Three of the four
protomers present in the asymmet-
FIGURE 4. Overall structure of T42SFlK in complex with AcCoA. The two protomers of the T42SFlK dimer are
represented in green and orange colors, respectively. A, 2F Ϫ 2F electron density map shows that AcCoA is
bound between two protomers of the active FlK dimer. B, AcCoA is sandwiched between the long ␤-sheet and
two small ␣-helices formed by residues 17–42. The long ␤-sheet and two small ␣-helices of protomer A and B
are shown in light blue and light purple, respectively. C, representation of the molecular surface shows that only
the acetyl and ␤-mercaptoethylamine moieties from AcCoA are buried in the active site of the protein.
o
c
from attack by solvent molecules. Based on our structural ric unit have a bound AcCoA, and in one of them, it was possible
evidence, we now propose that the catalytic triad in FlK com- to fit the complete molecule, despite the poor electron density
4
2
76
prises Thr -His -water (Fig. 2A), located at the interface for the 3Ј-phosphoryl-ADP moiety, possibly due to the high
between two protomers with two active sites in each FlK flexibility in the solvent. The overall structure of FlK does not
dimer (Fig. 2B).
change significantly as a result of the T42S point mutation and
4
Substrate Binding Pocket—Although attempts to co-crystal- the binding of AcCoA, but the AcCoA has forced Wat and
5
lize FlK with FAcCoA, CoA, or pantothenic acid failed, we Wat out of the active site cavity. The acetyl methyl group of the
5
successfully obtained crystals of T42SFlK with bound AcCoA AcCoA occupies the position of Wat , and the thioester car-
4
2
and WtFlK in complex with two analogues of fluoroacetyl-pan- bonyl oxygen interacts via a hydrogen bond to Ser -N. The
tetheine, FAcCPan, and FAcOPan. The sulfur atom of the thio- orientations of the three bound AcCoA molecules at the active
ester is replaced by a methylene in FAcCPan and by an oxy- site are significantly different, reflecting a dynamic AcCoA-FlK
gen atom in FAcOPan (Fig. 3). Comparison of the electron interaction process and the flexibility of the substrate binding
2
2500 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of Fluoroacetyl-CoA Thioesterase
6
9
and/or Gly -N in a seemingly ran-
dom manner in terms of hydrogen
bond donor-acceptor matching,
suggesting that the backbone amide
4
2
69
groups of Thr and Gly may be
crucial binding interactions avail-
able at the active site to bind the
substrate.
Computational
Docking
of
FAcCoA to FlK—We have modeled
a FAcCoA molecule into WtFlK
(
Fig. 5), based on the crystal struc-
FIGURE 5. Proposed FAcCoA coordination at the active site of FlK. A, a stick model with FAcCoA shown in tures of FlK in complex with various
silver and residues from the two protomers represented in yellow and green colors, respectively. B, a scheme
with FAcCoA (red) and active site residues (black). Hydrogen bonding interactions are denoted by dotted lines
with distance in Å.
ligands, especially with AcCoA. The
minimum energy was calculated
using the SYBYL 8.1.1.09097 pro-
gram. In a similar way to the AcCoA bound in T42SFlK, the
3
Ј-phosphoryl-ADP region of the docked FAcCoA is at the
enzyme surface in front of the entrance of the tunnel leading to
the active site. The rest of the molecule extends into the tunnel,
where the pantetheinyl moiety interacts through its N , O
1
2
9,
N , and C , respectively, with the main chain oxygen and the
1
4
19
7
6
79
128
side chain of His , Ala -N, and Phe -C . The hydrogen
⑀
1
1
20
50
bond between the side chains of Arg and Glu is broken due
5
0
FIGURE 6. Structural differences observed in the catalytic site of FlK due to the flip of the carboxyl group of the Glu . The fluorine is
4
2
42
76
to different mutations of Thr . The active site residues Thr , His , and
69
50
placed at a hydrogen bonding distance from Gly -N and the
Glu of WtFIK are superimposed with their counterpart residues in T42AFIK
1
20
120
120
4
2
76
50
and T42SFIK. A, Thr/Ala/Ser and His from protomer I and the Glu from guanidinium group of Arg . Mutation of the Arg to Ala
4
2
76
50
protomer II; B, Thr/Ala/Ser and His from protomer II and the Glu from
protomer I. Pink, WtFlK; green, T42AFlK; white, T42SFlK. The alternative con-
yielded a completely insoluble protein (data not shown), con-
120
7
6
sistent with the involvement of Arg in maintaining the con-
formations of His in T42A (pale green) and in T42S (yellow) are indicated.
4
2
formation of FlK. Thr -O␥ is now located closer (3.79 Å) to the
site. The acetyl carbonyl carbons of the three bound AcCoA thioester carbonyl carbon and at a better, although not optimal,
4
2
molecules are all in the wrong orientation with respect to O of position for a possible nucleophilic attack. The Thr -N that is
␥
42
Ser to allow nucleophilic attack. This explains the inability of close (2.81 Å) to the thioester carbonyl oxygen could act as an
FlK to hydrolyze AcCoA and implies that the presence of the oxyanion hole for stabilization of the tetrahedral acyl-enzyme
fluorine in FAcCoA is crucial for substrate recognition.
The positions of FAcCPan and FAcOPan in WtFlK differ
intermediate (Fig. 5).
Increased Flexibility at the Active Site Resulting from Muta-
42
42
significantly from that of AcCoA in the T42SFlK⅐AcCoA struc- tions of Thr —The hydrophobic interactions between Thr -
7
2
ture. Compared with the bound AcCoA, the bound FAcCPan is C and Ile -C in the adjacent protomer provide a “dry” envi-
␥2
␥2
4
2
buried deeper in the active site, and the FAcOPan is located ronment around the catalytic O of Thr , protecting it from
␥
1
closer to the entrance of the tunnel (Fig. 3B). In a way that attacks by solvent molecules, a feature observed for other
resembles the binding of AcCoA in T42SFlK, the carbonyl car- enzymes that have similar catalytic triad mechanisms (37). The
4
2
bons of both FAcCPan and FAcOPan in WtFlK are in the wrong Thr -C may also restrain the freedom of the substrate in the
␥
2
position and are even further away (3.88, 3.94, and 5.26 Å, active site. Loss of this restraint imposed by the C in the T42S
␥
2
42
respectively) from the O of Thr . Furthermore, the binding mutant may allow random misbinding of the substrate, which
␥
coordinates of the same molecule also vary in different FlK in turn may result in the substrate inhibition observed in the
dimers, suggesting that the presence of the nucleotide part of enzyme assays. In support of this notion, the imidazole group of
7
6
the CoA moiety is required for the correct overall engagement the His in the complex T42AFlK⅐FAc has a rotational disorder
of the substrate at the substrate-binding site.
of ϳ90° or exists in two alternative conformers not observed in
42
In all of the FAc or Ac-bound FlK complexes, more than one WtFlK structures, and the Ser in one of the protomers of the
molecule of FAc or Ac is bound in the large cavity of each active T42SFlK⅐Ac structure shows a double conformation, demon-
site. This may be a consequence of the high ligand concentra- strating the increased flexibility of these residues in the absence
4
2
tions (0.2 M) used in the crystallization solution and of the of the Thr -C and/or -O (Fig. 6).
␥
2
␥2
absence of the substrate. Interestingly but not surprisingly, at
Conformational Change for Product Release—The positions
each active site, there is always one ligand situated at a position of the main chains are almost completely conserved in all FlK
to where either the acetyl group of the AcCoA or the equivalent structures with or without mutations and/or bound ligands.
moiety of FAcCPan or FAcOPan is bound. The negatively Local conformational changes, especially in the hydrophobic
3
3
37
charged carboxylic oxygen moieties of the ligands and the loop FAEFP , are observed. The side chain conformations of
4
2
33
36
37
fluorine in the case of FAc are usually in contact with Thr -N Phe , Phe , and Pro in the structures with bound Ac or FAc
JULY 16, 2010•VOLUME 285•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 22501

Crystal Structure of Fluoroacetyl-CoA Thioesterase
bonding between the imidazole ring and the conserved water
molecule. Reformation of the carbonyl double bond breaks
the carbon-sulfur bond. A second tetrahedral intermediate is
then formed between the -OH part of a water molecule and
the carbonyl group of the fluoroacetyl-FlK intermediate. The
7
6
remaining proton of the water is bonded to His . When the
4
2
carbonyl bond reforms, the bond to the Thr -O is broken,
␥
7
6
releasing the fluoroacetate. The His -bonded hydrogen is
4
2
transferred to Thr -O , reestablishing the hydrogen bond-
␥
4
2
76
50
ing between Thr and His . The Glu , which is conserved
in most type II thioesterases as the catalytic residue, is not a
part of the catalytic mechanism of FlK but plays an impor-
tant role in maintaining the configuration of the active site.
It is not unexpected that T42SFlK is active because the
4
2
Ser -O␥ can still form a hydrogen bond with the imidazole
7
6
side chain of His and therefore has the potential to act as a
3
3
37
FIGURE 7. Representation of the hydrophobic loop FAEFP present in
the active site of FlK. Shown are the differences in the side chain for residues
4
2
nucleophile as the Thr -O in WtFlK. When FAcCoA con-
␥
33
36
37
Phe , Phe , and Pro for WtFlK⅐FAc (yellow) and apo-WtFlK (green).
centration was higher than 12 ␮M, however, excess-substrate
inhibition (38) was observed for the mutant. In other words,
differ from those of unbound WtFlK and the structures in com-
plex with AcCoA, FAcCPan, or FAcOPan (Fig. 7). In the
high concentrations of FAcCoA inhibited the activity of
42
T42SFlK. We speculate that the absence of the Thr -C in
␥
3
3
unbound WtFlK, the planes of the phenyl rings of Phe and
T42SFlK may have created more space, allowing an easier
access of substrate to the active site, especially at low sub-
36
Phe are nearly perpendicular to each other resembling a
closed two-door gate blocking the “exit” surrounded by the
major helix (the “sausage”), the ␤-sheet (the “bun”), and the
strate concentrations, giving rise to the lower K value and
m
the higher turnover of T42SFlK reaction as compared with
WtFlK activity. However, the same mutation also increased
protein flexibility, as shown by the double confirmations
of the active site residues in some of the protomers of the
mutant (Fig. 6). The enzyme-substrate complex formed be-
tween these abnormal active sites and substrate could be non-
productive. At high substrate concentrations, the chance
of non-productive enzyme-substrate complex formation
increases when more and more normal active sites are occupied
by FAcCoA. The formation of non-productive enzyme-sub-
strate complex at the active site of one protomer may also inter-
fere with the catalytic activity of the partner protomer of the
same dimer. These may explain the excess-substrate inhibition
phenomenon observed for T42SFlK and E50AFlK, but the exact
mechanism remains to be elucidated.
3
3
37
FAEFP loop. Binding of the substrate analogues, AcCoA,
FAcCPan, or FAcOPan, does not lead to any significant
change in the positions of the “doors.” When the active site is
occupied by the product FAc or its analogue Ac, the “gate” in
one of the protomers is wide open mainly due to a dramatic
3
6
swing and flip of the side chain of Phe , and the gate in the
other protomer of the same dimer is also open although to a
lesser extent. It is tempting to speculate that this opened gate
is the exit for the product FAc. The negatively charged car-
5
0
boxyl group of Glu , which has been “freed” from the hydro-
1
20
gen bond with the Arg side chain due to substrate binding,
is located near the gate and may facilitate expulsion of the
negatively charged FAc from the active site.
DISCUSSION
FlK is a homodimer with a hot dog fold similar to other
type II thioesterases, although it does not show extensive
sequence similarity to any type II thioesterases with known
function. A common feature shared by nearly all functionally
characterized hot dog fold thioesterases is the presence of an
acidic residue, aspartate or glutamate, providing general
base catalysis. One exception is FcoT, a long-chain fatty acyl-
CoA thioesterase from Mycobacterium tuberculosis, which
has a hot dog fold but uses an active site completely different
from that of either type I or type II thioesterases. FcoT has
been proposed to represent a new class of thioesterases, type
FlK, a thioesterase from fluoroacetate producing S. cat-
tleya, hydrolyzes the thioester bond of FAcCoA but not that
of AcCoA. The selectivity of FlK to distinguish FAcCoA from
AcCoA is remarkable because it ensures that AcCoA, a key
intermediate of the primary metabolism is not cleaved by
mistake. The only difference between the two compounds
is the presence of a fluorine atom in FAcCoA but not in
AcCoA. A combination of x-ray crystallography with site-
directed mutagenesis experiments allowed us to gain in-
sights into both catalytic mechanism and the unique sub-
strate specificity of FlK.
Our investigations suggest that FlK utilizes a catalytic triad III thioesterases (39). With a type II thioesterase fold, the
4
2
76
composed of a threonine (Thr ), a histidine (His ), and a catalytic machinery of FlK (Thr-His-water) resembles those
7
6
water molecule. The imidazole side chain of His acts as a of type I thioesterases (Ser/Cys-base-acid), exhibiting an
4
2
base to deprotonate the hydroxyl of Thr . The nucleophile unconventional combination of protein fold and catalytic
4
2
Thr -O attacks the fluoroacetyl carbonyl carbon, forming mechanism. Superposition of FlK with 18 structures of type
␥
a tetrahedral transition intermediate. The positive charge II thioesterases from the Protein Data Bank reveals that the
generated on the His76 residue is stabilized by the hydrogen Glu in FlK is well conserved in most of these thioesterases,
50
2
2502 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of Fluoroacetyl-CoA Thioesterase
120
69
although in some cases, it is an aspartate instead of a gluta- between the fluorine and Arg /Gly also help to stabilize this
mate (data not shown).
positive charge, making the carbonyl carbon a better target for
42
Among all of the hot dog fold proteins in the Protein Data nucleophilic attack by Thr -O (Fig. 5).
␥
Bank, only two hypothetical proteins, TTHA0967 (Protein
Data Bank entry 2CWZ) from Thermus thermophilus and Acknowledgment—We thank Dr. David O’Hagan (University of St.
Andrews) for helpful discussions.
TM0581 (Protein Data Bank entry 2Q78) from Thermotoga
maritime, have shown head-to-tail sequence similarity to FlK
(
33 and 22% identity, respectively). Superposition of FlK with
REFERENCES
these two structures has revealed a very similar putative active
site in 2CWZ and 2Q78. Most of the residues lining the active
1
. Sanada, M., Miyano, T., Iwadare, S., Williamson, J. M., Arison, B. H.,
Smith, J. L., Douglas, A. W., Liesch, J. M., and Inamine, E. (1986) J. Anti-
biot. 39, 259–265
42
50
69
76
site of FlK, Thr , Glu , Gly , and His , are well conserved in
36
44
63
76
38
2
CWZ (Thr , Glu , Gly , and His ) and in 2Q78 (Thr ,
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His , and Arg ) with the following differences. First, the
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3. Schaffrath, C., Deng, H., and O’Hagan, D. (2003) FEBS Lett. 547,
counterpart of the Arg
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115
120
111–114
(
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4
5
6
7
8
. Deng, H., O’Hagan, D., and Schaffrath, C. (2004) Nat. Prod. Rep. 21,
5
0
guanidinium group with the carboxyl side chain of Glu . The
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amide side chain of Gln in CWZ2, however, is pointing in the
opposite direction with no possibility of forming any direct
. Dong, C., Huang, F., Deng, H., Schaffrath, C., Spencer, J. B., O’Hagan, D.,
and Naismith, J. H. (2004) Nature 427, 561–565
44
. Zhu, X., Robinson, D. A., McEwan, A. R., O’Hagan, D., and Naismith, J. H.
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putative active site of 2CWZ due to this change of Arg to Gln. If
(
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. Peters, R., and Wakelin, R. W. (1953) Proc. R. Soc. Lond. B Biol. Sci. 140,
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2
CWZ is also an acyl-CoA thioesterase, the substrate should
have a larger acyl moiety than a fluoroacetyl group. Second, the
50
69
46
65
equivalents of FlK Glu and Gly in 2Q78 are His and Val ,
46
respectively. The imidazole side chain of the His and the iso-
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65
(
1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13699–13703
propyl group of the Val at the active site provide a binding
motif quite different from that in FlK.
1
0. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M.,
Harel, M., Remington, S. J., Silman, I., Schrag, J., Sussman, J. L., Vers-
chueren, K. H. G., and Goldman, A. (1992) Protein Eng. 5, 197–211
1. Pleiss, J., Fischer, M., and Schmid, R. D. (1998) Chem. Phys. Lipids 93,
67–80
The significant differences in the overall positions of
FAcCPan and FAcOPan in the active site of FlK as compared
with that of the acetyl-pantheteinyl segment of the T42SFlK-
1
bound AcCoA (Fig. 3) suggest that binding of the nucleotide 12. Leesong, M., Henderson, B. S., Gillig, J. R., Schwab, J. M., and Smith, J. L.
(
1996) Structure 4, 253–264
portion of the substrate on the enzyme surface dictates the
depth to which the fluoroacetyl pantetheinyl arm extends
into the active site cleft, ensuring the correct interaction
between the arm with the amino acid residues in the vicinity
of the active site. Although the sulfur atom of the thioester is
1
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JOURNAL OF BIOLOGICAL CHEMISTRY 22503

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2504 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 29•JULY 16, 2010

Protein Structure and Folding:
Structural Basis for the Activity and
Substrate Specificity of Fluoroacetyl-CoA
Thioesterase FlK
Marcio V. B. Dias, Fanglu Huang, Dimitri Y.
Chirgadze, Manuela Tosin, Dieter Spiteller,
Emily F. V. Dry, Peter F. Leadlay, Jonathan
B. Spencer and Tom L. Blundell
J. Biol. Chem. 2010, 285:22495-22504.
doi: 10.1074/jbc.M110.107177 originally published online April 29, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M110.107177
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