(Received for publication, September 11, 1995)
From the
On the basis of the sequence similarity between mammalian
epoxide hydrolases and bacterial haloalkane dehalogenase reported
earlier (Arand, M., Grant, D. F., Beetham, J. K., Friedberg, T., Oesch,
F., and Hammock, B. D.(1994) FEBS Lett. 338, 251-256;
Beetham, J. K., Grant, D., Arand, M., Garbarino, J., Kiyosue, T.,
Pinot, F., Oesch, F., Belknap, W. R., Shinozaki, K., and Hammock, B.
D.(1995) DNA Cell. Biol. 14, 61-71) we selected
candidate amino acid residues for the putative catalytic triad of the
rat soluble epoxide hydrolase. The predicted amino acid residues were
exchanged by site-directed mutagenesis of the epoxide hydrolase cDNA,
followed by the expression of the respective mutant enzymes in Escherichia coli. A total of 25 different mutants were
analyzed for their epoxide hydrolase activity toward the model
substrate trans-stilbene oxide. In case of impaired catalytic
activity of a given mutant, the structural integrity of the recombinant
enzyme protein was assessed either by its ability to covalently bind
the substrate trans-stilbene oxide or by affinity purification
on benzyl thio-Sepharose, using the soluble epoxide hydrolase-specific
competitive inhibitor 4-fluorochalcone oxide to release the bound
enzyme from the affinity matrix. Of the mutants under investigation,
only those with changes in the positions Asp,
Asp
, and His
were completely inactive
toward the model substrate trans-stilbene oxide while
retaining the proper protein fold. These amino acids were exactly those
previously predicted by sequence alignment. Exchange of the amino acid
residues flanking the catalytic nucleophile Asp
significantly changed the kinetic properties of the enzyme.
Mutation of His
to Gln had no apparent effect on the K
but led to a heavily reduced V
(5% that of the wild type) of the mutant
enzyme, while the exchange of Trp
against Phe strongly
increased the K
(7-fold) and also
moderately enhanced the V
(2-fold) of the
corresponding mutant. Mutation of Trp
apparently had a
strong effect on the protein conformation.
Epoxide hydrolases (EH; ()EC 3.3.2.3) are a group of
functionally related enzymes that catalyze the addition of water to
oxirane compounds (epoxides), thereby generating vicinal trans-diols(1) . (
)This enzymatic reaction
represents a detoxification step of central importance, because (i)
many oxirane derivatives are reactive electrophiles, due to the high
tension of the three-membered ring system and the strong polarization
of the C-O bonds, and (ii) epoxides are frequent intermediary
metabolites arising during the biotransformation of foreign
compounds(2) . EH have been found in all types of living
organisms, including mammals, invertebrates, plants, fungi, and
bacteria and may thus be regarded as ubiquitous. In mammals, the two EH
implicated in the xenobiotic metabolism are the microsomal epoxide
hydrolase (mEH) (3) and the soluble epoxide hydrolase
(sEH)(4) . These two enzymes are distinct by substrate
specificity, subcellular distribution and inducibility by foreign
compounds(5, 6) .
Very recently, much progress has
been made in understanding the structural relationship between these
two mammalian EH. They share amino acid sequence similarity to a region
around the active center of the bacterial haloalkane
dehalogenase(7) , an enzyme with a known three-dimensional
structure that belongs to the family of /
hydrolase fold
enzymes(8) . This led us and others to hypothesize that EHs
also belong to the
/
hydrolase fold
family(9, 10, 11) . The common feature of
this family of proteins is a highly conserved protein fold in the
so-called domain I, composed of a central sheet of parallel
-strands that is sandwiched by
-helices (12) (see Fig. 1). This domain provides the framework for a catalytic
triad, similar to that of serine proteases, with which these enzymes
perform their catalytic task. The catalytic mechanism involves two
steps: in a first reaction, an ester bond is formed between enzyme and
substrate by attack of the substrate with a nucleophilic amino acid
residue (Ser, Cys, or Asp; see N in Fig. 1), and this
ester is subsequently hydrolyzed in a second step by a water molecule
that has been activated via proton abstraction by a His-Asp/Glu pair (H and A in Fig. 1). The reaction mechanism
for the sEH, as inferred from the analogy to that of haloalkane
dehalogenase(13) , is shown in Fig. 2. First
experimental evidence for the correctness of this proposed mechanism
was brought about by Lacourciere and Armstrong for the mEH (14) and Hammock et al. for the sEH (15) .
Lacourciere and Armstrong showed that rat mEH incorporates
O during substrate turnover in H
O
and is subsequently capable of transferring this
O into
the product of the enzymatic reaction in the complete absence of
H
O. The stoichiometry of the reaction is best
explained by the intermediate formation of an enzyme-substrate ester.
Hammock et al. could unequivocally demonstrate intermediate
covalent binding of the substrate juvenile hormone III to murine sEH by
precipitation of the enzyme-substrate ester and subsequent
microchemical analysis.
Figure 1:
General three-dimensional structure of
/
hydrolase fold enzymes. This is a schematic representation
of the structurally conserved domain I of
/
hydrolase fold
enzymes as described by Ollis et al.(12) .
-Helices are shown as spirals, and
-strands are
symbolized by flat arrows. Helices and strands are marked with letters and numbers, respectively, according to (12) . The important elements of the active center are as
follows: nucleophile (Ser, Cys, or Asp) (N); water-activating
histidine (H); and acidic residue of the charge relay system
(Asp or Glu) (A). Domain II is not structurally conserved
among all
/
hydrolase fold enzymes, but it is present between
strand 6 and helix D in the haloalkane dehalogenase (8) and the
bromoperoxidase(34) , the two
/
hydrolase fold
enzymes of solved three-dimensional structure with sequence similarity
to epoxide hydrolases.
Figure 2:
Reaction mechanism of soluble epoxide
hydrolase. Epoxide hydrolysis by sEH proceeds by a mechanism analogous
to the dechlorination by haloalkane dehalogenase(13) . In step
1 of the enzymatic reaction, the reactive nucleophile Asp attacks the oxirane ring at one of the carbon atoms, leading to a
ring opening and the formation of an enzyme-substrate ester. This
covalent intermediate is hydrolyzed in step 2 of the reaction by a
water molecule that is activated via proton abstraction by the
His
-Asp
pair. The role of the amino acid
residues specified by the index numbers in the catalytic process is
suggested by sequence comparison to related proteins (see Fig. 3) and verified by the results of the present
report.
Figure 3:
Alignment of important conserved amino
acid sequence elements of selected sEH-related proteins. Amino acid
sequences are given in the one-letter code. The positions of the
residues involved in the catalytic mechanism are marked with arrows. Asterisks indicate residues in the sEH
sequence that were mutated in the present study. Numbers in italics designate the position of the adjacent amino acid in the
respective sequence. Numbers between back slashes represent
the number of connecting amino acid residues not shown in the
alignment. Numbers in brackets represent the total number of
amino acids of the respective protein. In case of sequence identity
50% among the 10 sequences at a given position, the common amino
acid residue is boxed in the respective sequences and printed
in the consensus, with the exception of the catalytic nucleophile that
is marked by a plus sign in the consensus. Note that sequence
identity is never below 30% in the displayed range. The horizontal
bars at the bottom of the diagram represent the position
of the reported secondary structural elements of haloalkane
dehalogenase (13) and bromoperoxidase(34) . sEH, rat soluble epoxide hydrolase(17) ; mEH,
rat microsomal epoxide hydrolase(37) , aEH, soluble
epoxide hydrolase from Arabidopsis thaliana(33) ; pEH, soluble epoxide hydrolase from Solanum
tuberosum(32) ; HALO, haloalkane dehalogenase
from Xanthobacter autotrophicus GJ10 (7) ; BPH-RP, human biphenyl hydrolase-related protein(38) ; BPH D, biphenyl hydrolase from P. putida KF715 (39) ; DMPD, 2-hydroxymuconic semialdehyde hydrolase
from P. putida CF600(40) ; XYLF,
2-hydroxymuconic semialdehyde hydrolase from P.
putida(41) ; BPA2, bromoperoxidase A2 from S.
aureofaciens(34) ; Con, consensus sequence; N, nucleophile; H, water-activating histidine; A, acidic residue of the charge relay
system.
The aim of the present study was to provide
experimental proof for the composition of the catalytic triad of rat
soluble epoxide hydrolase as predicted by sequence similarity analysis
(for a condensed sequence alignment, see Fig. 3). Our strategy
was to substitute the amino acid residues in question by site-directed
mutagenesis and to analyze the resulting recombinant proteins after
expression in bacteria for their catalytic activities and their
structural integrity. Those mutants with lost enzymatic activity but
apparently retained structural conformation should be directly involved
in the catalytic mechanism. While this study was underway, Pinot et
al.(16) reported that the mutation of murine sEH at
positions Asp and His
leads to inactive
enzyme and that these residues, therefore, are part of the catalytic
triad of sEH, corresponding to N and H in Fig. 1. In the present report we identify Asp
as
the third member (A) of the catalytic triad of the rat sEH, in
addition to confirm the function of Asp
and His
also in the rat enzyme. Furthermore, we describe altered kinetic
properties of mutant rat sEH due to the exchange of amino acids
neighboring the catalytic nucleophile Asp
.
Figure 4:
Structure of the vector constructs for the
recombinant expression of mutant sEH. The schematic representation of
the vector pRSET B-sEH highlights important features for the expression
of the recombinant sEH proteins. The gene for the sEH, represented as a dark arrow in the vector map, is additionally given
in more detail to point out the positions of amino acids that are
exchanged in the different mutants and the restriction enzyme
recognition sites used for the construction of the respective
expression vector derivatives. The putative domain structure of sEH is
indicated by respective segmentation of the bar representing
the protein coding sequence. The vector-derived His-tag fusion part of
the recombinant protein is followed by the N-terminal HAD1-like domain
of sEH (1, 35) of yet obscure function, which possibly
contains a second active center. This is connected to the domain I of
the postulated /
hydrolase fold of the
sEH(9, 10, 11) , which is interrupted by the
domain II and therefore split into domain Ia and domain Ib in the figure. 3`-UTR designates the noncoding part of the sEH cDNA
within the expression construct.
Figure 5:
Enzymatic activity of the sEH mutants. The bars represent the enzymatic activity of the mutant sEH
proteins toward trans-stilbene oxide in relation to the wild
type recombinant enzyme. Wild type activity was 325 ± 48 nmol of
substrate converted per min and mg of sEH-protein, as judged by the
immunoquantification outlined under ``Experimental
Procedures.'' The error bars indicate the standard
deviation obtained by at least three independent expressions. The
mutated amino acid residues are specified by indexing the one-letter
code symbol with the number indicating its position in the protein
sequence. The corresponding amino acids resulting from the mutation
process are given without index. n.d., not detectable
(0.3% of wild type activity); *, chain termination (stop
codon).
The
expression of the different sEH constructs in E. coli JM109
resulted in the massive production of respective fusion proteins in the
bacteria. The non-EH part of the recombinant proteins was a
vector-derived N-terminal peptide of 41 amino acids, containing a
(His)-tag to facilitate purification of the recombinant
proteins by immobilized metal affinity chromatography. Immunoblot
analysis of the expression products in the bacterial 13,000
g supernatants using purified native rat sEH as a standard
revealed the accumulation of immunoreactive protein with an apparent
molecular mass of 67 kDa, which corresponds to the expected increase in
molecular mass of about 5 kDa as compared with the native enzyme. The
amount of soluble recombinant protein obtained was remarkably constant
within a range of 5-10 mg of sEH protein expressed per liter of
culture as determined by quantification of the immunosignals. Little
clone-to-clone variation was observed, with the exception of the mutant
with the inverted MscI-fragment, which expressed at a
3-5-fold lower level. Despite serious attempts we were unable to
purify the fusion proteins by immobilized metal affinity
chromatography.
Figure 6: Coprecipitation of radioactive substrate with sEH mutant protein. The bars represent the amount of radioactive substrate that coprecipitates with the recombinant proteins under the conditions described under ``Experimental Procedures.'' The mutant constructed by inverting the MscI fragment in the sEH coding sequence was used as the negative control in that the amount of radioactivity coprecipitated with it (166 ± 77 cpm) was subtracted as the blank from the values obtained for the other proteins under investigation. Only mutants with low or undetectable enzymatic activity were analyzed. The error bars indicate the standard deviation obtained by at least three independent experiments. The mutated amino acid residues are specified by indexing the one-letter code symbol with the number indicating its position in the protein sequence. The corresponding amino acids resulting from the mutation process are given without index. WT, wild type recombinant sEH; Y*, H517Y/C521Y double mutant (not shown in Fig. 5); N*, H517N/H523N double mutant (not shown in Fig. 5).
Based on sequence
alignments (1, 9, 11) (Fig. 3),
Asp is predicted to be the nucleophile that is directly
involved in the ester formation between the enzyme and the substrate
during the first step of the enzymatic reaction (Fig. 2).
Therefore, a mutant enzyme with an exchange of Asp
against a nonacidic amino acid may not be expected to covalently
bind the substrate. To prove the structural integrity of the
Asp
mutants we tested their capability to bind to benzyl
thio-Sepharose, the affinity matrix used for the specific retention of
sEH in a one-step purification procedure(26) . Both the D333G
and the D333C mutants were specifically retained by the affinity matrix
and eluted like the wild type enzyme when the washing buffer (75 mM Tris-Cl, pH 7.4) was fortified with 0.5 mM 4-fluorochalcone oxide, a potent competitive inhibitor of sEH,
while the mutant with the inverted MscI fragment did not bind
to benzyl thio-Sepharose. However, while the Gly mutant behaved
indistinguishably from the wild type, significantly less protein of the
Cys mutant was retained by the affinity material in parallel
experiments.
Figure 7: Kinetic analysis of selected sEH mutants. Lineweaver-Burke representation of the substrate dependence of the catalytic activity of the sEH mutants W334F (A), H332Q (B), and W540L (B), in comparison with that of the wild type recombinant enzyme (A). Note the different scales in A and B. WT, wild type recombinant sEH.
The recent advances in molecular cloning of soluble epoxide
hydrolases from several different mammalian (17, 29, 30, 31) and plant (32, 33) species have significantly enhanced the
understanding of the relationship between the different epoxide
hydrolases and other proteins with structural similarity. The
similarity of the C-terminal part of sEH and mEH to the bacterial
haloalkane dehalogenase led us to postulate that EH as well as a number
of other enzymes that share this sequence similarity belong to the
/
hydrolase fold family of enzymes(9) .
Interestingly, the three-dimensional structure for one of these other
enzymes, namely the bromoperoxidase A2 from Streptomyces
aureofaciens, has recently been elucidated and confirmed the
assignment of the enzyme to the
/
hydrolase fold family that
we had previously predicted(34) . By similarity analysis (1, 9, 10, 11) , Asp
could be almost unequivocally identified as the nucleophile
necessary for the first step of the sEH-mediated epoxide hydrolysis.
The role of His
for the water activation during the
second step of catalysis was strongly suggested by the exceptionally
high conservation between the amino acid sequence around His
in sEH and the corresponding region in the hydroxymuconic
semialdehyde hydrolases from Pseudomonas putida and, to a
somewhat lesser extent, the haloalkane dehalogenase(9) . The
prediction of the third member of the potential catalytic triad by
sequence comparison was more ambiguous because of a lower degree of
sequence conservation around the respective acidic residue. However,
two independent reports both came to the conclusion that Asp
was the best candidate for this catalytic
residue(1, 11) . Still obscure is the function of the
N-terminal sEH sequence that is 230 residues in length and shows
significant similarity to bacterial haloacid
dehalogenases(1, 35) . In the present study, we
investigated the catalytic properties of mutants that were specifically
designed to prove or disprove the role of the above named amino acid
residues in the catalytic process. In addition to the identified
candidate residues we exchanged additional amino acids that were
possible alternatives for the former, although of much lower
probability. Of the five acidic residues we exchanged individually,
three turned out to be without major importance for the catalytic
activity, namely Glu
, Asp
, and
Glu
, while substitution of the two amino acids predicted
to be part of the catalytic triad, Asp
and
Asp
, resulted in completely inactive mutants. Covalent
substrate binding in the case of Asp
His and the
capability to bind to the affinity purification matrix benzyl
thio-Sepharose in the case of Asp
Gly and
Asp
Cys demonstrated that the loss of enzymatic activity
was not due to an improper folding of the proteins but must be
explained by the direct involvement of the two mutated residues in the
catalytic mechanism. Three histidines were substituted: the major
candidate His
, His
as the one being most
proximal to it, and His
that directly precedes the
catalytic nucleophile Asp
. The three mutants with an
exchanged His
were all enzymatically inactive, and two of
them, H523N and H523Y, were able to covalently bind the substrate trans-stilbene oxide. The third mutant, H523D, did not
coprecipitate any substrate, most likely due to a loss of structural
integrity. This is readily explained by the potential electrostatic
repulsion taking place by replacing the basic histidine, that is
sandwiched in the catalytic triad between two aspartic acids, with a
third acidic residue. While the mutant H517D retained about 50% of the
enzymatic activity as compared with the wild type, two His
double mutants were enzymatically inactive. The one with an
additional C521Y mutation could not covalently bind the substrate,
while the other one with the additional mutation H523N did. These
findings were perfectly in line with our expectations, because complete
loss of activity was only associated with retained structure, as judged
by substrate binding, in the case of a His
mutation. The
third histidine exchange, H332Q, resulted in a strongly impaired yet
detectable enzymatic activity of the resulting mutant. Neither the K
nor the ability to covalently bind the substrate
was impaired for the mutant as compared with the wild type, which
indicates that the second step of the enzymatic reaction, i.e. the ester hydrolysis, must be affected by the exchange.
Trp
, the other amino acid residue flanking the
nucleophile of the catalytic triad, is highly conserved among the
epoxide hydrolases and the haloalkane dehalogenase. In the latter
enzyme, a role of this tryptophan in hydrogen bonding the chloride
released from 1,2-dichloroethane by the haloalkane dehalogenase has
been reported, and we have hypothesized that this tryptophan might, in
analogy, support the opening of the epoxide ring by sEH via hydrogen
bonding the oxirane oxygen. However, exchange of Trp
to
phenylalanine did not reduce the enzymatic activity but enhanced K
by a factor of 7 and V
by
a factor of 2. A similar result was recently reported for the analogous
mutation of haloalkane dehalogenase(36) . We conclude that
Trp
is not mechanistically involved in the catalytic
process of epoxide hydrolysis but is of some importance for the binding
of the substrate.
Our first attempt to align the amino acid
sequences of mEH and sEH suggested a small motif near the carboxyl
terminus of both EH, namely the tetrapeptide
Lys-Trp-Leu-Lys/Lys-Trp-Val-Lys corresponding to positions
539-542/411-414 of sEH/mEH, respectively. ()Thus, Lys
, Trp
, and
Lys
were the first positions where we constructed
mutants. Exchange of either of the lysine residues against arginine or
methionine did not affect the enzymatic activity to a great extent. On
the other hand, the mutation of the tryptophan had a strong effect.
Exchange against leucine or serine strongly reduced the enzymatic
activity, and introducing a stop codon at this position completely
abolished the catalytic activity. In contrast, a frameshift mutation
leading to a different amino acid sequence from position 545 on had no
apparent effect on the enzymatic activity. Neither of the Trp
mutants displayed a detectable extent of covalent substrate
binding. On the other hand, the apparent K
had
decreased rather than increased with the mutant W540L. Sequence
alignment (Fig. 3) offers a possible explanation for these
observations. Trp
is amino acid number 17 C-terminal to
the catalytic histidine. At the corresponding position, all other
sequences of the alignment have a highly conserved phenylalanine. This
position is at the end of the last
-helix of the two sequences of
known three-dimensional structure and is oriented toward the
-sheet. The behavior of the Trp mutants is consistent with a role
of the conserved aromatic amino acid residue in fixing helix F with the
catalytic histidine His
close to its N terminus (see Fig. 3) in a position to retain the proper conformation of the
active center. A fraction of 5% of the mutant proteins being in the
active conformation would not have been picked up by our substrate
binding assay, due to the relatively low sensitivity of this analytical
procedure.
In conclusion, our findings demonstrate that (i) the
catalytic triad of rat sEH is composed of the residues Asp (the nucleophile), Asp
(the histidine-supporting
acid), and His
(the water-activating histidine) (ii) the
amino acid residues flanking the nucleophile significantly influence
kinetic parameters of the enzymatic reaction, and (iii) Trp
has an important function in stabilizing the structure of the
enzyme.
This paper is dedicated to Prof. Dr. Dr. Ernst Mutschler, on the occasion of his 65th birthday.