From the Laboratory of Biophysical Chemistry and
BIOSON Research Institute and the § Laboratory of
Biochemistry, Department of Chemistry, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epoxide hydrolases catalyze the
cofactor-independent hydrolysis of reactive and toxic epoxides. They
play an essential role in the detoxification of various xenobiotics in
higher organisms and in the bacterial degradation of several
environmental pollutants. The first x-ray structure of one of these,
from Agrobacterium radiobacter AD1, has been determined by
isomorphous replacement at 2.1-Å resolution. The enzyme shows a
two-domain structure with the core having the Epoxide hydrolases (EC 3.3.2.3) are a group of functionally
related enzymes that catalyze the cofactor-independent hydrolysis of
epoxides to their corresponding diols by the addition of a water
molecule. Epoxides are very reactive electrophilic compounds frequently
found as intermediates in the catabolic pathway of various xenobiotics.
For instance they are the carcinogens formed by bioactivation reactions
catalyzed by cytochrome P450. Therefore, conversion of epoxides to less
toxic, water-soluble compounds is an essential detoxification step in
living cells. Consequently, epoxide hydrolases have been found in a
wide variety of organisms, including mammals, invertebrates, plants,
and bacteria (1).
Until now most research has been focused on mammalian epoxide
hydrolases (2, 3), which, together with glutathione
S-transferases, are the most important enzymes to convert
toxic epoxides to more polar and easily excretable compounds (4).
However, much progress has recently also been made in the
characterization of bacterial epoxide hydrolases (5, 6, 7). These
enzymes show a significant sequence homology with those of mammalian
origin. They can be easily obtained in large amounts, and they exhibit
enantioselectivity with various industrially important epoxides, which
makes them promising biocatalysts for the large scale preparation of
enantiopure epoxides and/or their corresponding vicinal diols (8). In
particular, extensive studies have been performed on the epoxide
hydrolase from Agrobacterium radiobacter AD1, a
Gram-negative bacterium that is able to use the environmental pollutant
epichlorohydrin as its sole carbon and energy source (5, 6, 8). This epoxide hydrolase is a soluble monomeric globular protein of 35 kDa
with a broad substrate range. Epichlorohydrin and epibromohydrin are
its best substrates, and the optimum pH range for catalysis is
8.4-9.0. Sequence and secondary structure analysis suggested that this
enzyme belongs to the Here we report the 2.1-Å resolution x-ray structure of the epoxide
hydrolase from A. radiobacter AD1
(Ephy).1 It is the first
epoxide hydrolase for which the structure has been solved. The result
of this work can provide a general better understanding about the
structural basis of the reaction mechanism for this class of important
ubiquitous enzymes.
Crystallization and Heavy Atom Search--
The epoxide
hydrolase from A. radiobacter AD1 was cloned, overexpressed,
and purified as described previously (6). The stock protein solution,
containing 5 mM potassium phosphate, 1 mM EDTA,
1 mM
The crystals diffract up to 2.1-Å resolution using synchrotron
radiation, and they belong to space group C2 with unit cell parameters of a = 146.62 Å, b = 100.20 Å, c = 96.88 Å, Data Collection and Processing--
A 2.1-Å resolution native
data set and one of the two ethyl mercury phosphate derivative data
sets were collected at the x-ray diffraction beamline of the ELETTRA
synchrotron in Trieste (Italy), equipped with a 30-cm MAR image plate
area detector (MAR Research, Hamburg, Germany) with the wavelength
tuned to Structure Determination and Refinement--
The structure of the
epoxide hydrolase from A. radiobacter AD1 was solved by the
method of single isomorphous replacement supplemented by anomalous
scattering, using both the in-house and synchrotron derivative data
sets. A major heavy atom site (8.5
The model was traced using the program O (18). Nearly the complete
polypeptide chain of one monomer could be interpreted in agreement with
the amino acid sequence. By applying the refined NCS operators to the
coordinates of the first molecule, coordinates for the other three
molecules in the asymmetric unit were generated. The four molecules
were then refined using the program X-PLOR (19). During the first runs
of the refinement (simulated annealing and individual B-factor
refinement), tight NCS restraints were applied (17), but in the final
stage of the refinement (conventional positional refinement and
individual B-factor refinement), they were gradually released or not
even used at all for those residues that clearly showed different
conformations in the 4 monomers in the asymmetric unit. The best
refinement results were obtained using a flat bulk solvent correction.
Special care was taken in the selection of the test set for the
Rfree calculation; the test set was selected by
dividing the reflections in 102 thin-resolution shells to minimize the
correlation between test set and working set reflections that could be
caused by the presence of NCS (20). Water molecules were placed
according to strict density and distance criteria, starting with the
buried and NCS-related ones.
The final model consists of 4 × 282 residues, 610 water
molecules (33 of them refined with double positions), and 4 potassium ions. The crystallographic R factor and
Rfree are 19.0% and 22.7%, respectively.
PROCHECK (21) and WHATCHECK (22) were used to assess the stereochemical
quality. The structure was further analyzed using the program VOIDOO
(23), the programs from the CCP4 suite (14), the BIOMOL package, and
the program DALI (24). Refinement statistics are given in Table
II. The atomic coordinates and the structure factors have been deposited to the Protein Data Bank with the
entry code 1ehy.
Modeling of Asp246--
As a starting model, the
atomic coordinates of the refined structure of the wild type epoxide
hydrolase were used in which only the internal solvent molecules were
retained. The crystal structure was energy-minimized prior to the
modeling using a conjugate gradient routine implemented in X-PLOR (19).
To completely remove the possible bias because of the conformation of
the protein in the crystal, a slow-cooling molecular dynamics
simulation (19) of 25 ps with temperature coupling (25) was performed
in which the temperature was slowly reduced from 1000 K to 300 K. The
missing loop 138-148 and the loop containing Asp246 were
built using the program O (18).
To model a likely conformation of the active Ephy enzyme, we
assumed that the acid member of the catalytic triad,
Asp246, should lie at interaction distance to the catalytic
His275 side chain, as found in many other members of the
Several cycles of stereochemical regularization were performed using
the REFI and LEGO option of O (18). The model was subsequently subjected to energy minimization to tidy up unacceptable close contacts
and poor stereochemistry. To overcome the possibility that the
energy-minimized structure was trapped in a local minimum, a second
molecular dynamics-simulated annealing run was performed using the same
setting as before. Extensive energy minimization was applied until
convergence was reached, leading to a model with no residues outside
the allowed regions in the Ramachandran plot (29) and good
stereochemical quality (r.m.s. deviation bond lengths = 0.005 Å,
r.m.s. deviation bond angles = 1.61°). Asp107 and
Asp131 have slightly deviating backbone torsion angles,
like in the x-ray structure.
Although the position of the modeled loop 132-148 is only one of the
possible conformations it can assume, we are confident that the
rebuilding of the loop containing Asp246, in a fashion
common to many Quality of the Model--
Epoxide hydrolase from A. radiobacter AD1 (Ephy) crystallizes in the monoclinic space group
C2, with 4 molecules in the asymmetric unit. A
superimposition of the C Overall Structure--
The Ephy monomer has a nearly globular
shape with approximate dimensions of 48 × 47 × 47 Å3. It consists of two domains: domain I (or "core"
domain), which shows the typical features of the Active Site--
The proposed active-site residues (6)
Asp107 and His275 are located in a
predominantly hydrophobic internal cavity between domains I and II. The
core domain contributes to the lining of the cavity with residues
Gly37, Trp38, Pro39,
Glu44, His106, Asp107,
Phe108, Ile133, Phe137,
Ile219, His275, Phe276,
Val279. The cap domain supplies Tyr152,
Trp183, and Tyr215 (Fig.
3).
Asp107 is situated at the very sharp "nucleophile
elbow" between the central strand
An ~20-Å long tunnel, filled with water molecules, is located
between
Asp246 has been proposed to be the acidic member of the
catalytic triad, responsible for assisting His275 in
activating the water molecule that hydrolyzes the ester intermediate formed at Asp107 (6). Asp246 is located in a
turn between strand Comparison with
Despite a low sequence homology (33% homology, 20% identity), the
structural similarity of epoxide hydrolase and haloalkane dehalogenase
is particularly interesting. These enzymes have both an Asp-His-Asp
catalytic triad. Asp107 and His275 of Ephy
superimpose very well on Asp124 and His289 of
DhlA; their side chains are in the same relative position and make
similar hydrogen bonds. In dehalogenase the halogen atom of the
substrate is bound between the indole ring N-atoms of
Trp125 and Trp175. In epoxide hydrolase,
Phe108 and Trp183 occupy the equivalent
positions, suggesting that they may be involved in substrate binding
(6). However, a stabilizing structural role for Phe108 is
also conceivable, as it has a "T-shaped" interaction with the
Tyr215 side chain (31). In dehalogenase, the O Modeling of Asp246--
As mentioned above, the third
catalytic residue, Asp246, is pulled out of the active
site. However, the positions of Asp260 in haloalkane
dehalogenase and Asp228 in bromoperoxidase A2 give a
reliable suggestion where Asp246 should be located in the
active conformation of Ephy. Superposition of the
The final model shows an intact and empty active site cavity,
capable of accommodating substrates. It is lined with
Gly37, Trp38, Pro39,
Glu44, His106, Asp107,
Phe108, Ile133, Phe137,
Tyr152, Trp183, Tyr215,
Ile219, Cys248, His275,
Phe276, and Val279 (Fig.
7). Asp246 is hydrogen-bonded
to the His275 N Active Site and Substrate Binding--
Epoxide hydrolase has a
two-domain structure (Fig. 1). The core domain displays an
The third member of the catalytic triad, Asp246, assists
His275 in activating the hydrolytic water molecule (6). To
our surprise it is not at hydrogen bonding distance from the N
In the crystal structure the Gln134 side chain oxygen is
hydrogen-bonded to the hydroxyl group of Tyr152 and
Tyr215 (Fig. 3). These two tyrosines are the only acidic
functional groups present in the active site that can facilitate the
opening of the epoxide ring by hydrogen bonding and protonating the
epoxide oxygen. In agreement with this hypothesis, mutagenesis studies of these tyrosines have shown that only a double Tyr-Phe mutant is
completely inactive, suggesting that both Tyr152 and
Tyr215 are able to provide the proton needed for the
opening of the epoxide ring.4
Because Tyr215 is absolutely conserved within the epoxide
hydrolase family and Tyr152 is mostly conserved in the
soluble epoxide hydrolases, it is likely that the Tyr activation is a
general property of this class of enzymes (Fig. 8). In the past, one of
three lysines, Lys173, Lys174, and
Lys177, was proposed to be involved in the protonation
(6), but the crystal structure of Ephy unambiguously shows that these
three lysine residues are located far from the active site, exposed to
the solvent on top of the cap domain.
Position and Function of Asp131--
The
Asp246
Asp131 is at an equivalent position to Asn148
in haloalkane dehalogenase. In the dehalogenase, Asn148 is
involved in a hydrogen-bonding stabilization of the active site (10,
26, 30). Like Asn148, Asp131 in Ephy also shows
slightly unusual backbone torsion angles. Furthermore, in the
dehalogenase, the activity of an Asp260
A similar shift of functional residues has been observed for
Pseudomonas glumae lipase (Pgl) (34) and Hpl (35), two other members of the Oxyanion Hole--
A conserved HGXP tetrapeptide motif
(X = any amino acid) is found in epoxide hydrolases and
other
In haloalkane dehalogenase, this sequence motif is HGEP, and the main
chain N atom of the glutamate residue (Glu56) is, together
with the peptide nitrogen atom of residue Trp125, part of
the oxyanion hole, interacting with the O
A second role of the tetrapeptide motif may be in stabilizing the
position of the putative hydrolytic water molecule (Fig. 6). Indeed, in
the crystal structure this water molecule is at interacting distance to
the backbone oxygen atom of Trp38.
These essential structural functions may explain the importance of the
HGXP motif for enzymatic activity within the epoxide hydrolase family, as already demonstrated by mutation of His to Ala in
the rat microsomal epoxide hydrolase (37).
Conclusions--
The x-ray structure reveals for the first time
the fold of an epoxide hydrolase and provides novel, detailed
information on the residues involved in the enzymatic mechanism. It
localizes the catalytic residues, the hydrolytic water molecule, and
the position of the oxyanion hole, and it proposes a possible backup for the acidic member of the catalytic triad. Most importantly, it
unambiguously identifies the previously unanticipated
Tyr152/Tyr215 as the acidic group responsible
for binding and possibly protonation of the transition state of the
formation of the ester intermediate. The residues important for
catalysis are conserved within the epoxide hydrolase family. Therefore
all these structural features are likely to be shared by other epoxide
hydrolases and allow us to gain a better understanding of the behavior
and mechanism of this class of biologically and biotechnologically
important enzymes. At present we are investigating the structural basis of the enzymatic enantioselectivity by mutation analysis and by docking
the substrates in the modeled active site.
/
hydrolase-fold
topology. The catalytic residues, Asp107 and
His275, are located in a predominantly hydrophobic
environment between the two domains. A tunnel connects the back of the
active-site cavity with the surface of the enzyme and provides access
to the active site for the catalytic water molecule, which in the
crystal structure, has been found at hydrogen bond distance to
His275. Because of a crystallographic contact, the active
site has become accessible for the Gln134 side chain, which
occupies a position mimicking a bound substrate. The structure suggests
Tyr152/Tyr215 as the residues involved in
substrate binding, stabilization of the transition state, and possibly
protonation of the epoxide oxygen.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
hydrolase-fold family of enzymes (9).
Site-specific mutations indicated Asp107,
His275, and Asp246 as the catalytic triad
residues. The proposed catalytic mechanism involves two steps analogous
to haloalkane dehalogenase (10). In the first reaction step, an ester
bond is formed between enzyme and substrate by attack of the
nucleophilic Asp107 on the primary carbon atom of the
substrate; in the second step, this ester bond is hydrolyzed by a water
molecule activated by the His275/Asp246 pair.
The reaction proceeds via two different transition states, one during
the binding and opening of the epoxide ring and the second during the
hydrolysis of the ester intermediate. However, several important
questions remained unanswered. Until now it has not been possible to
identify the residue responsible for the binding and protonation of the
epoxide oxygen, nor was the location known of the oxyanion hole that
stabilizes the Asp107 oxyanion during the hydrolysis of the
ester intermediate. Structural information may also resolve why an
Asp246
Ala mutant still retains some residual activity
(6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 0.02% sodium azide, and 10%
glycerol (pH 6.8) was concentrated and extensively washed using 10 mM potassium phosphate (pH 7.0) in a Centricon-10
ultracentrifugation concentrator with a 10-kDa cut-off (Amicon) to a
final protein concentration of 5.5 mg/ml. The extensive washing
procedure is essential to remove the glycerol before crystallization.
The glycerol induces high polydispersity in the protein sample, as was
determined by dynamic light scattering analysis on a DynaPro 801 instrument (Protein Solutions, Charlottesville, VA). Removing the
glycerol resulted in a solution containing particles with a 2.7-nm
diameter (apparent molecular mass of 33 kDa) and a polydispersity of
15%. Crystallization experiments with the protein in the presence of glycerol gave only very thin needles as a best result. An initial promising crystallization condition was obtained from a Sparse Matrix
screening (11). Refinement of this Sparse Matrix condition resulted in
the following crystallization protocol: hanging drops (4 µl of
protein solution and 4 µl of precipitant) were equilibrated against a
1-ml reservoir containing 1.6 to 1.8 M
KH2PO4/K2HPO4 (pH 7.0)
at room temperature. After 2 weeks, the experiments were allowed to
slowly evaporate to a phosphate concentration of about 2.0 M. The slow increase of the phosphate concentration in the drop results in the appearance of crystals with typical sizes of
0.3 × 0.2 × 0.1 mm3. They are highly
x-ray-sensitive, and therefore, all data collections were performed at
cryotemperature (100 K), using 30% glycerol added to the stabilizing
mother liquor (1.8 M
KH2PO4/K2HPO4) as a cryoprotectant.
= 100.68°. This unit cell gives
a VM value of 2.57 Å3/Da
1
assuming 4 molecules in the asymmetric unit. The deduced solvent content of the crystals is 52%. Heavy atom derivatives were prepared by soaking the crystals in solutions obtained by dissolving the heavy
atom compounds in the standard mother liquor (1.8 M
KH2PO4/K2HPO4). The
search resulted in only one good isomorphous derivative obtained by
soaking a crystal of epoxide hydrolase for 2 days in a solution of 2.0 mM ethyl mercury phosphate,
(C2H5HgO)3PO.
= 1.0 Å. An in-house derivative data set was collected on
a Mac Science DIP-2030H area detector equipped with a dual 30-cm image
plate, with graphite monochromatized CuK
radiation (
= 1.5418 Å)
from a FR591 rotating anode generator with a double mirror x-ray
focusing system (model MAC-XOS) as x-ray source (Enraf Nonius, Delft,
The Netherlands). All data sets were collected at 100 K, integrated,
and merged using the DENZO/SCALEPACK package (12) and software from the BIOMOL crystallographic package (Protein Crystallography Group, University of Groningen). Derivative data were scaled to the native data set using the program PHASES (13). Data-processing statistics are
given in Table I.
) for the ethyl mercury phosphate
derivative was located in a difference Patterson map (12.0-4.5-Å
data). The remaining 21 heavy atom positions were determined using
difference Fourier techniques. Heavy atom position search, parameter
refinement including anomalous data, and phase calculations were
performed with PHASES (13) (Table I). The
initial phases calculated at 3.7 Å yielded a figure of merit of 0.47 and were improved by solvent flattening and histogram matching
techniques using the program DM (14). The noncrystallographic symmetry
(NCS) operators (three orthogonal 2-fold axes) relating the 4 molecules
in the asymmetric unit were determined with the help of
FINDNCS,2 using the 8 heavy
atom sites with the highest occupancies. They were checked by comparing
them with the rotation matrices calculated from a self-rotation
function (16). An initial mask was built around one molecule in the
asymmetric unit with the program MAMA (17); this mask was then used to
refine the NCS operators by maximizing the correlation between the
electron density maps of the 4 molecules in the asymmetric unit using
the program IMP (17). Iterative cycles of density averaging,
improvement of the mask, and refinement of the NCS operators, along
with solvent flattening and phase extension to 2.6 Å resolution,
resulted in a map of interpretable quality.
Data collection and single isomorphous replacement including anomalous
scattering (SIRAS) analysis
Refinement statistics and stereochemical quality of the final model
/
hydrolase-fold family (9). Haloalkane dehalogenase (PDB
accession code 2HAD) (26) and bromoperoxidase A2 (PDB accession code
1BRO) (27) were used as templates to model the new Ephy
Asp246 position, analogous to Asp260 of
dehalogenase and Asp228 of bromoperoxidase, respectively.
Secondly, we assumed that the Gln134 side chain should be
removed from the active site, as it blocks the putative substrate
binding site. This was done by giving the Pro132-Ile133-Gln134 loop a similar
conformation as the human pancreatic lipase (PDB accession code 1LPB)
(28) Pro177-Ala178-Glu179 motif,
which has an equivalent topological position. The loop of residues
138-148, which is not observed in the electron density, was built like
in the bromoperoxidase structure, connecting the core and the cap domains.
/
hydrolase-fold enzymes, gives a plausible
picture of the catalytic site of the fully active epoxide hydrolase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
atoms of the four molecules gave an average
r.m.s. difference of 0.24 Å for molecules B, C, and D and a higher
r.m.s. difference of 0.40 Å if molecule A is included. All results
discussed below apply to all 4 molecules (A, B, C, and D) unless stated
otherwise. The final model consists of 4 × 282 residues, 610 water molecules (33 of them refined with double positions), and 4 potassium ions, originating from the crystallization buffer, one for
each molecule in the asymmetric unit. In each monomer (294 residues),
the first N-terminal residue (Met) is not visible nor is there
interpretable electron density for the loop 138-148. The final
crystallographic R factor and Rfree
values are 19.0% and 22.7%, respectively. The r.m.s. deviations from
ideal geometry are 0.008 Å for bond lengths and 1.338° for bond
angles. No residues are in the disallowed regions of the Ramachandran
plot (29). Pro39 was found in a cis conformation.
/
hydrolase-fold
topology (9), and the mainly
-helical domain II (or "cap"
domain), which lies on top of domain I (Figs.
1 and 2).
The core domain comprises amino acids 1-137 and 219-294, and it
consists of a central eight-stranded
-sheet with seven parallel
strands (only the second strand is antiparallel). The
-sheet is
flanked on both sides by
-helices, two on one side and four on the
other. Helix
9 is a one-turn 310 helix. Domain II,
containing
-helices
4 to
8, forms a large excursion between
-strands
6 and
7 of the core domain. It has a double-layered
structure with helices
7 and
8 located between the core domain
and the plane formed by
4,
5, and
6.
View larger version (79K):
[in a new window]
Fig. 1.
Schematic view of the secondary structure
elements (from DSSP (38)) of the epoxide hydrolase
monomer. Ribbon representation drawn using MOLSCRIPT (39) is
shown; -helices,
-strands, and coils are represented by
helical ribbons, arrows, and ropes,
respectively. The
-helices of the cap domain are shown in dark
gray.
View larger version (27K):
[in a new window]
Fig. 2.
Secondary structure topology diagram and
location of the catalytic triad residues, Asp107,
Asp246, and His275. The dashed
line represents the missing loop 138-148. Short 310
helices are located at the N terminus ( '), between
3 and
1
(
'1), between
4 and
2 (
'2), and between
3 and
6
(
'3). The last
-helix shows a conspicuous bend at residue 281 (
'11-
11) because of the presence of Pro282 in the
center of the helix.
View larger version (26K):
[in a new window]
Fig. 3.
Stereo view of residues lining the active
site of epoxide hydrolase. Residues and water molecules are drawn
in ball and stick representation using MOLSCRIPT (39). The
catalytic water molecule is labeled WAT.
5 and helix
3. At this
topological position, all
/
hydrolase-fold enzymes present the
nucleophile, which can either be Ser, Cys, or Asp (9). The (
,
)
angles of Asp107 are slightly unfavorable (
= 57°,
=
124°), but its conformation is stabilized by a network of
hydrogen bonds involving residues of the sharp turn, as has been found
in other
/
hydrolase enzymes (9). In addition, the main chain
nitrogen atom of Asp107 interacts via a hydrogen bond with
the backbone oxygen atom of Asp131, the other residue with
slightly deviating backbone torsion angles (
= 31°,
= 69°).
Furthermore, the side chain of Asp107 is stabilized by a
hydrogen bond of its O
2 atom with the backbone amide groups of
Trp38 and Phe108 and by a salt bridge between
the O
1 atom of Asp107 and the N
2 atom of the
His275 side chain.
-helices
1,
10, the loop connecting
-helix
1 and
-strand
3 of the core domain, and
7 of the cap domain (Fig. 4). This tunnel leads to the back of the
active-site cavity, and it is perfectly suited to replenish the
hydrolytic water molecule at hydrogen bond distance to the N
2 atom
of the His275 side chain (Fig. 3) after the reaction. In
our structure, the active site cavity is exposed to the solvent from
the front part too, where the missing loop is located. Because of the
position of the hydrolytic water molecule in the back of the active
site, it is likely that the substrate enters the active-site cavity from the front part.
View larger version (33K):
[in a new window]
Fig. 4.
Stereo view of the tunnel connecting the back
of the active site with the protein surface. The tunnel was
calculated with VOIDOO (23) using a probe with radius 1.2 Å. The core
and the cap domain are shown in light and dark
gray, respectively. Side chains of Asp107 and
His275 are in ball and stick representation. The
figure was drawn using BOBSCRIPT (15).
7 and helix
10, in a position topologically
conserved within the
/
hydrolase-fold family (Fig. 2) (9).
However, in our crystal structure Asp246 is not at
interacting distance from His275. Instead the loop
containing this residue is pulled away from the active site, and the
Asp246 side chain is pointing into the solvent. This is
probably the result of crystal packing forces because helix
10 of
molecule A, which follows the loop containing Asp246, is
involved in an intermolecular contact with helix
10 of molecule B. A
similar contact exists between molecules C and D, which even involves
an intermolecular disulfide bridge between Cys(C)248 and
Cys(D)248. The absence of this disulfide bond between
molecules A and B results in a slightly different conformation of the
loop containing Cys248 in molecule A compared with the
other three molecules in the asymmetric unit. As a consequence, the
difference between the C
positions of molecules A, B, C, and D
(average r.m.s. difference of 0.40 Å) is higher than for the B, C, and
D molecules only (r.m.s. difference of 0.24 Å). The conformational
plasticity of the region between residues 244 and 257 is also reflected
by very high B-factors (between 28 Å2 and 84 Å2) and a not easily interpretable electron density map,
often poorly defined or showing multiple conformations even for the
backbone. The space vacated by Asp246 makes it possible for
the side chain of Gln134 to move into the active site,
occupying the site where the substrate is likely to be bound (Fig. 3).
Its position is stabilized by a hydrogen bonding network involving the
hydroxyl groups of Tyr152 and Tyr215 and the
carboxyl oxygen O
1 of Asp107.
/
Hydrolases--
The folding of Ephy
strongly resembles that of bromoperoxidase A2 from Streptomyces
aureofaciens (27) (BpA2, PDB accession code 1BRO; r.m.s. deviation
~1.7 Å for 193 C
atoms) and haloalkane dehalogenase from
Xanthobacter autotrophicus (10, 26, 30) (DhlA, PDB accession
code 2HAD; r.m.s. deviation ~ 2.0 Å for 204 C
atoms) and a
number of other members of the
/
hydrolase-fold family (9). The
matching is best for the central
-sheet and for helices
2 and
3 (Fig. 5), but all other structural
elements are equivalent as well, especially in the regions close to the catalytic residues. The
-helices in the cap domain superimpose less
well, showing a different relative orientation. These helices contribute several residues important for the interaction with substrates.
View larger version (23K):
[in a new window]
Fig. 5.
Superimposition of the cores of epoxide
hydrolase from A. radiobacter AD1
(black), haloalkane dehalogenase from X. autotrophicus (white), and bromoperoxidase
A2 from S. aureofaciens (gray).
A, superimposition of the central -sheet. B,
superimposition of the region around the position of the nucleophile,
with
-strand 5,
-helix 3, and
-strand 6. The figure was
produced using MOLSCRIPT (39).
2 atom of
Asp124, which in the putative transition state will become
negatively charged, is stabilized by interaction with the main chain
nitrogen atoms of residues 125 and 56. Asp107 O
2 in Ephy
has similar interactions with the amide nitrogen atoms of residues 108 and 38, suggesting Phe108 and Trp38 as part of
the oxyanion hole.
-sheets of DhlA
and BpA2 on that of Ephy brings the O
2 atom of Asp260 of
dehalogenase and Asp228 of bromoperoxidase in coincidence
with the water molecule in Ephy, which is hydrogen-bonded to N
1 of
His275 and O
2 of Asp131 (Fig.
6). This information was used to model a
likely conformation of the "active" Ephy enzyme, with DhlA, BpA2,
and human pancreatic lipase (Hpl) structures as templates for
reconstructing the Ephy loops containing Asp246 and
Gln134 (See "Experimental Procedures").
View larger version (41K):
[in a new window]
Fig. 6.
Stereo view of the water-mediated interaction
between Asp131 and His275 and of the oxyanion
hole in epoxide hydrolase. Hydrogen bonds are shown as
dashed lines. Side chains, water molecules, and the HGWP
motif are in ball and stick representation. The catalytic
water molecule is labeled WAT. The figure was produced using
MOLSCRIPT (39).
1, and it now occupies the position where
the acidic member of the catalytic triad is normally found in
/
hydrolase-fold enzymes. The rest of the active site has undergone only
minor changes in the relative positions of the atoms. The hydrolytic water molecule is still present at hydrogen bond distance to the His275 N
2 atom. The Tyr152 and
Tyr215 hydroxyl groups, which in the crystal structure were
hydrogen-bonded to the Gln134 side chain, still point in
the same direction, enabling them to donate the proton needed for
opening of the epoxide ring (Fig. 8).
View larger version (45K):
[in a new window]
Fig. 7.
Stereo view of the active site of the model
of epoxide hydrolase. Side chains and water molecules are in
ball and stick representation. The catalytic water molecule
is labeled WAT. The figure was drawn using MOLSCRIPT
(39).
View larger version (19K):
[in a new window]
Fig. 8.
Schematic representation of the catalytic
mechanism of epoxide hydrolase. The Michaelis complex with
epichlorohydrin is shown before the formation of covalent intermediate,
which is indicated by arrows. Hydrogen bonds are
shown as dashed lines.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
hydrolase-fold topology, which provides the scaffolding for the
catalytic triad residues Asp107, His275, and
Asp246, whereas the
-helical cap domain contributes
several residues important for the interaction with substrates. The
active site is located in a cavity between the two domains, which
contains Asp107 and His275. Asp107
is the nucleophile that attacks the substrate carbon atom in the first
step of the reaction, whereas His275 activates the water
molecule that hydrolyzes the ester intermediate in the second reaction
step (6). Indeed, in the crystal structure, a water molecule is visible
at hydrogen bond distance to the N
2 atom of the His275
side chain. Because of the crystallization pH (7.0), in the x-ray structure, His275 is stabilized by a salt bridge with
Asp107 O
1. At the optimum pH for catalysis (8.4-9.0)
His275 N
2 is most likely deprotonated and therefore able
to activate the water molecule located nearby. In the crystal
structure, this water molecule is in contact with the solvent via a
narrow tunnel between the core and cap domains (Fig. 4). This tunnel
resembles the active site back entrance in bromoperoxidase A2, which
was proposed to provide access to the active site for small molecules participating in the reaction, like peroxide and halide, or to expel
water molecules from the active site during substrate binding (27). In
epoxide hydrolase, it seems more likely that the tunnel serves to
replenish the hydrolytic water molecule after the reaction has been completed.
1 atom
of His275, but it has moved away into the solvent region.
This is most probably a consequence of crystal packing forces: the
Asp246
Ala mutation strongly decreases the activity of
the enzyme (~0.5% of the wild type activity) (6), and such a
dramatic effect on activity is difficult to rationalize for the
position of the residue as observed in our crystals. The exposed
position of Asp246 has made it possible for the
Gln134 side chain to move into the active site and block it
(Fig. 3). Because a Gln134
Ala mutant has an activity
comparable with that of wild-type enzyme, it is unlikely that
Gln134 is normally present in the active site. However,
human microsomal epoxide hydrolase has been reported to be inhibited by
amides (32). The A. radiobacter epoxide hydrolase shows
competitive inhibition by amides as well, especially by compounds like
phenylacetamide (Ki = 30 µM).3
Therefore, we conclude that the Gln134 side chain may act
as such an inhibitor, mimicking the binding mode of epoxide substrates.
Thus, the combination of crystal contacts of helix
10 (residues 252 to 261) and the affinity of the active site for amide compounds have
probably led to the observed exposed position of Asp246.
Nevertheless, the high structural similarity (Fig. 5) between the core
domains of epoxide hydrolase, haloalkane dehalogenase, bromoperoxidase
A2, and human pancreatic lipase allowed us to use the latter three
enzyme structures as templates to remodel the loops containing
Asp246 and Gln134. The result is a plausible
model of the active site site in the fully active enzyme (Fig. 7).
Ala mutation resulted in a strong reduction of
enzymatic activity. Nevertheless, this mutant still has some residual activity (~0.5% that of wild type activity), indicating the
importance of Asp246 in catalysis but not its essentiality
(6). The three-dimensional structure of Ephy shows the presence of
another aspartic acid, Asp131, which may act as a backup of
Asp246 (Fig. 3). Asp131 is located between
strand
6 and the first helix of the cap domain, with its side chain
in close contact with the imidazole ring of the catalytic
His275. A water molecule, present in all four monomers in
the asymmetric unit, bridges the interaction between O
2 of
Asp131 and N
1 of His275 (Fig. 6).
Asn mutant
could be restored by an Asn148
Asp/Glu mutation. This
shows that the catalytic triad in DhlA can be either
Asp-His-Asp260 or Asp-His-Asp148 (33). The same
could be true for Ephy, where Asp131 could take over the
function of Asp246 by a simple rotation around the
1 and
2 torsion angles, which would allow its side chain to be
hydrogen-bonded to His275 N
1.
/
hydrolase-fold family. In Pgl,
Glu288 can take over the role of Asp263 as the
acid residue of the catalytic triad. Glu288 O
2 and
Asp263 O
2 of Pgl match the positions of
Asp131 O
2 and of its hydrogen-bonded water molecule in
Ephy. In human pancreatic lipase, an alternative catalytic triad is
present in which the acid catalytic residue is shifted from
-strand
7 to
-strand 6 at position 176. Asp176 of Hpl overlaps
with Asp131 in Ephy, with the Asp176 O
2 atom
matching the position of the water molecule hydrogen-bonded to
Asp131 in Ephy. These observations make Asp131
a very interesting residue for site-specific mutational studies to
further probe its role in catalysis.
/
hydrolase-fold enzymes (2, 3). The HGWP motif of Ephy is
located in a sharp cis-proline turn
(Trp38-Pro39), which is stabilized by the
hydrogen bond between His36 N
1 and the backbone carbonyl
oxygen of Gly37 (Fig. 6).
2 atom the nucleophile
Asp124 (10, 26). In epoxide hydrolase, a similar hydrogen
bonding pattern is present between the O
2 atom of the nucleophile
Asp107 and the amide nitrogen atoms of Trp38
and Phe108. Thus these peptide nitrogen atoms are in an
optimal position to stabilize the negative charge that develops on the
O
2 atom of the nucleophile during the hydrolysis of the ester
intermediate. In addition, the negative charge on Asp107
O
2 may be further stabilized by the
-helix dipole of helix
3
(36).
![]() |
ACKNOWLEDGEMENT |
---|
We thank A. Savoia and the staff of the x-ray diffraction beamline at the ELETTRA synchrotron, Trieste (I), for the synchrotron data collection facilities and assistance.
![]() |
FOOTNOTES |
---|
* Financial support was obtained from the European Union (Eu-lipase contract number BI02-CT94-3013). Work at the ELETTRA synchrotron was supported by the European Union, contract number ERBFMGECT950022.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (1ehy) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ To whom correspondence should be addressed. Fax: 31-50363-4800: E-mail: bauke{at}chem.rug.nl.
2 G. Lu, http://gamma.mbb.ki.se/~guoguang/findncs.html.
3 R. Rink and D. B. Janssen, manuscript in preparation.
4 R. Rink, J. H. Lutje Spelberg, R. J. Pieters, J. Kingma, M. Nardini, R. M. Kellogg, B. W. Dijkstra, and D. B. Janssen, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Ephy, epoxide hydrolase from A. radiobacter AD1; DhlA, haloalkane dehalogenase from X. autotrophicus GJ10; Pgl, triacylglycerol lipase from P. glumae; Hpl, human pancreatic lipase; NCS, noncrystallographic symmetry; r.m.s., root mean square.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|