Crystal Structures of Reaction Intermediates of
L-2-Haloacid Dehalogenase and Implications for the Reaction
Mechanism*
Yong-Fu
Li
,
Yasuo
Hata
§,
Tomomi
Fujii
,
Tamao
Hisano
¶,
Mitsuhiro
Nishihara
,
Tatsuo
Kurihara
, and
Nobuyoshi
Esaki
From the
Institute for Chemical Research, Kyoto
University, Uji, Kyoto 611-0011 and the ¶ Institute of
Physical and Chemical Research (RIKEN), Wako,
Saitama 351-0106, Japan
 |
ABSTRACT |
Crystal structures of
L-2-haloacid dehalogenase from Pseudomonas sp.
YL complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-methylbutyrate, or
L-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-Å resolutions, respectively, using the complex crystals
prepared with the S175A mutant, which are isomorphous with those of the
wild-type enzyme. These structures exhibit unique structural features
that correspond to those of the reaction intermediates. In each case,
the nucleophile Asp-10 is esterified with the dechlorinated moiety of
the substrate. The substrate moieties in all but the monochloroacetate
intermediate have a D-configuration at the C2
atom. The overall polypeptide fold of each of the intermediates is
similar to that of the wild-type enzyme. However, it is clear that the
Asp-10-Ser-20 region moves to the active site in all of the
intermediates, and the Tyr-91-Asp-102 and Leu-117-Arg-135 regions
make conformational changes in all but the monochloroacetate
intermediates. Ser-118 is located near the carboxyl group of the
substrate moiety; this residue probably serves as a binding residue for
the substrate carboxyl group. The hydrophobic pocket, which is
primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151,
Asn-177, and Trp-179 side chains, exists around the alkyl group of the
substrate moiety. This pocket may play an important role in stabilizing
the alkyl group of the substrate moiety through hydrophobic
interactions, and may also play a role in determining the
stereospecificity of the enzyme. Moreover, a water molecule, which is
absent in the substrate-free enzyme, is present in the vicinities of
the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177,
and Ala-175 in each intermediate. This water molecule may hydrolyze the
ester intermediate and its substrate. These findings
crystallographically demonstrate that the enzyme reaction proceeds
through the formation of an ester intermediate with the enzyme's
nucleophile Asp-10.
 |
INTRODUCTION |
L-2-Haloacid dehalogenase
(L-DEX)1 is a
unique enzyme that catalyzes the hydrolytic dehalogenation of
L-2-haloacids to produce the corresponding
D-2-hydroxyacids with an inversion of the
C2-configuration. Various L-DEXs exhibiting
very similar sequences have been isolated from different bacterial
sources. Of all the enzymes, L-DEX YL, isolated from a
2-chloroacrylate-utilizable bacterium, Pseudomonas sp. YL,
is an unusual L-DEX, in that it is relatively thermostable even though it is derived from a mesophilic bacterium (1). It is a
dimeric enzyme formed by two identical subunits of 232 amino acid
residues. We have already reported the crystallization of
L-DEX YL (2) and its crystal structure determined by a
2.5-Å resolution x-ray analysis (3). The enzyme has a core domain of
/
-structure, which differs topologically from those of the
/
hydrolase fold family proteins (4), along with a subdomain having a four-helix-bundle structure. Our ion spray mass spectrometric study showed that the dehalogenation of the L-2-haloacid
catalyzed by L-DEX YL proceeds in the two-step mechanism
through an ester intermediate (5). In the proposed reaction mechanism,
the nucleophile Asp-10 first attacks the C2 atom of the
substrate to form an ester intermediate and a halide ion, and,
subsequently, a water molecule hydrolyzes the intermediate by attacking
the C
atom of Asp-10 to produce a
D-2-hydroxyacid, restoring the side-chain carboxyl group of
Asp-10. This mechanism has been further investigated by mass spectral
analyses of the enzyme paracatalytically inactivated by hydroxylamine
(6); the enzyme forms an adduct of the dehalogenated substrate moiety
and hydroxylamine specifically at the Asp-10 position. We replaced each
of the charged or polar amino acid residues (total 36 residues) of
L-DEX YL, which are conserved in most of the
L-DEXs, with another residue and found that, in addition to
Asp-10, the residues of Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157,
Ser-175, Asn-177, and Asp-180 probably play an essential role in
catalysis (7). X-ray crystallography revealed that all the residues
except Arg-41 are located around the putative active site, nucleophile
Asp-10, and that nine water molecules in this region form a complicated
hydrogen bond network with the functionally important residues. All of
the residues are thought to be involved in the catalytic process, which
comprises selective recognition of the L-enantiomer of the
2-haloacid, binding of the carboxyl group of the substrate
L-2-haloacid, capture or withdrawal of the halide ion
liberated from the L-2-haloacid upon nucleophilic attack by
the side-chain carboxylate of Asp-10, formation of the ester
intermediate, and activation of a water molecule for hydrolysis.
The reactions catalyzed by haloalkane dehalogenase from
Xanthobacter autotrophicus GJ10 (8-10), rat liver
microsomal epoxide hydrolase (11), and 4-chlorobenzoyl-CoA
dehalogenases from Pseudomonas sp. strain CBS3 (12-14) and
Arthrobacter sp. 4-CB1 (15) also proceed through mechanisms
similar to that of L-DEX YL, which involve the formation of
an enzyme-substrate ester intermediate for which the acyl moiety is
derived from a carboxyl group of the enzyme. Haloalkane dehalogenase,
epoxide hydrolase, and 4-chlorobenzoyl-CoA dehalogenase each represent
individual families of proteins that differ from the L-DEX
family. The x-ray crystallographic studies of haloalkane dehalogenase
showed that the enzyme is a member of the
/
hydrolase fold
family, which includes acetylcholinesterase, carboxypeptidase, and
lipase (4). Members of the family have a common fold comprising an
eight-stranded parallel
-sheet flanked by
-helices, where the
catalytic triad residues are located at topologically equivalent
positions on particular turns and loops. In the haloalkane dehalogenase
(9, 10), Asp-124 carries out a nucleophilic attack on the
C1 atom of substrates such as 1,2-dichloroethane to give an
ester intermediate that is subsequently hydrolyzed by a water molecule
activated by His-289. The halogen atom released from the substrate is
captured by Trp-125 and Trp-175. In L-DEX YL, however, no
histidine residue is present in the putative active site. His-19, which
is the only histidine residue conserved of all L-DEXs,
is not essential for the catalysis of L-DEX YL (16). These
findings suggest that L-DEX YL utilizes a residue other than histidine to activate a water molecule for hydrolysis. Like the
haloalkane dehalogenase, a conserved tryptophan residue, Trp-179, is
located in the vicinity of the nucleophile Asp-10 in L-DEX YL, but it is not essential for the catalysis of the enzyme. The other
three conserved tryptophan residues, Trp-40, Trp-49, and Trp-193, are
located outside the active site. It is therefore assumed that in the
case of L-DEX YL, the abstraction of the halogen is
performed by residues other than tryptophan.
In order to elucidate the mechanism of catalysis and
stereoselectivity of L-DEX YL by identifying the amino acid
residues involved, it is indispensable to study tertiary structures of L-DEX YL complexed with its substrate in each step of the
catalysis. We crystallized several mutants of L-DEX YL that
are suitable for this purpose. Of these mutants, the S175A mutant
provided good crystals, which allowed us to perform a high resolution
x-ray crystallographic study. We prepared crystals of the S175A mutant complexed with various kinds of substrates and to analyze their crystal
structures by x-ray diffraction techniques. Four kinds of complexes
gave electron density maps that unequivocally showed the formation of
ester intermediates. Here we report the crystal structures of MCA, CBT,
CMB, and CMV intermediates at 2.2-1.83-Å resolutions. The structures
of these intermediates are essential not only for the identification of
amino acid residues participating in each step of the catalysis but
also for the elucidation of the mechanism of catalysis and
stereoselectivity of L-DEX YL.
 |
MATERIALS AND METHODS |
Preparation and Assay of Enzymes--
The wild-type and mutant
enzymes of L-DEX YL were overproduced in recombinant
Escherichia coli cells and purified by ammonium sulfate
fractionation followed by DEAE-Toyopearl chromatography (7, 17). The
mutants Y12F and S175A of L-DEX YL were prepared as
described previously (7), and the Y12A (GTA
GGC), Y12L (GTA
GAG), and Y12W (GTA
CCA) mutants
were prepared by the method of Kunkel (18), using the synthetic
mutagenic primers (Biologica, Nagoya, Japan).
The activity of the wild-type and mutant enzymes for halide release was
assayed with 25 mM MCA, CPA, CBT, CMB, and CMV as substrates. The reagents
DL-(RS)-2-chloro-n-butyrate,
L-(S)-2-chloro-3-methyl-n-butyrate, and
L-(S)-2-chloro-4-methyl-n-valerate
(Tokyo Kasei) were used as the substrates for CBT, CMB, and CMV,
respectively. Polyethylene glycol 400 was added to the reaction mixture
to a final concentration of 15% (v/v) to increase the solubility of
these reagents. The amount of chloride ion released from the substrate
was spectrophotometrically determined according to the method of
Iwasaki et al. (19). One enzyme unit of halide release
activity was defined as the amount of enzyme that releases 1 µmol of
halide ion/min. The amount of D-lactate produced from 25 mM L-2-chloropropionate was determined by
measuring the amount of NADH produced by the reaction of
D-lactate dehydrogenase with D-lactate in terms
of absorbance at 340 nm. In a typical assay, the reaction was started
at 30 °C by adding 0.141 nmol of the wild-type enzyme or 13.0 nmol
of the S175A mutant to a 1-ml reaction mixture of 0.1 M
Tris-H2SO4 solution (pH 9.5) containing 25 mM CPA and 5 mM NAD, and then 18.2 units of the D-lactate dehydrogenase (Sigma) were immediately added to
the solution. The enzyme activity producing D-lactate from
L-2-chloropropionate was estimated from the ratio of the
initial velocity to the molar absorption coefficient of 6,220 M
1 cm
1 for NADH; 1 unit was
defined as the amount of enzyme needed to produce 1 µM
NADH/min. The amount of enzyme was estimated with a Bio-Rad protein
assay kit. The activities of the wild-type enzyme and the S175A mutant
are listed in Table I. Our ion-spray mass spectrometric study suggests
that the production of D-hydroxyacid by hydrolysis after
the halogen abstraction is the rate-determining step in the
dehalogenation of L-2-haloacids catalyzed by
L-DEX YL.2 These
results suggest that the S175A mutant could be virtually inactive
toward MCA, CBT, CMB, and CMV in the crystal at pH 5.5, although the
mutant does display detectable activity toward these substrates in the
solution at optimum pH.
Crystal Preparation--
Crystals of the S175A mutant were grown
by a method similar to that for the wild-type enzyme reported
previously (2, 3). Rhombohedral crystals were obtained by the vapor
diffusion of a 15 mg/ml enzyme solution against a reservoir solution
(pH 5.5) consisting of 50 mM potassium dihydrogenphosphate,
15% (w/v) polyethylene glycol (approximate Mr
8000), and 1% (v/v) n-propanol at 4 °C. The typical
crystal size was approximately 0.6 mm × 0.5 mm × 0.2 mm.
The crystals belong to space group C2 with unit cell
dimensions of a = 92.47 Å, b = 62.78 Å, c = 50.99 Å, and
= 122.7°, contain one
subunit of the dimer molecule in the asymmetric unit, and are
isomorphous with those of the wild type. They are of good quality
suitable for high resolution x-ray analysis and diffract to better than
1.8-Å resolution.
Crystals of the S175A mutant complexed with each of the substrates MCA,
CBT, CMB, and CMV were prepared by a soaking method. All of the
substrates except MCA were dissolved in the reservoir solution
containing 15% polyethylene glycol 400. The S175A crystals soaked in
the reservoir solution containing 5 mM MCA diffracted well
and gave diffraction intensities significantly different from those of
the S175A crystals, which implied the formation of a complex. However,
most of the S175A crystals were dissolved within 10 min in the
reservoir solution with a concentration as low as 10 µM
CPA, and cracked in reservoir solutions containing 1 mM
CPA, CBT, CMB, or CMV. Therefore, a technique of cross-linking with
glutaraldehyde was applied to reinforce the crystals before soaking
them in the solutions containing each of the substrates except for MCA.
The cross-linking conditions for the crystals were investigated in
solutions containing 10 mM CBT, CMB, or CMV by changing the
concentration of glutaraldehyde and the soaking time. Finally, the
crystals were cross-linked in 5% (v/v) glutaraldehyde for 5 h at
4 °C. However, even the cross-linked crystals cracked in the 1 mM CPA solution. CPA is best among all the substrates of
the enzyme and has the lowest Km value (7). This is
the case with the S175A mutant, as shown in Table
I. Binding of CPA to the enzyme may
induce such large structural changes of the molecules in the crystal as
to destroy the crystal packing. Efforts to prepare CPA-complexed
crystals failed completely. Consequently, the complex with CPA was
excluded from the present analysis.
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Table I
Relative activities of wild type and S175A toward various substrates
Values shown are percent ratios to the wild-type activity in each step
against L-2-chloropropionate.
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The unit cell dimensions of all the complexed crystals are shown in
Table II. The crystal of the MCA complex
is isomorphous with that of the S175A mutant, although the
crystallographic c axis is shortened by 0.51 Å (1.0%).
However, the unit cell dimensions of the CBT-, CMB-, and CMV-complexed
crystals shrunk by about 1.1-1.5 Å (1.2-1.6%) and a maximum of 0.7 Å (1.3%) in the directions of the crystallographic a and
c axes, respectively. These phenomena indicate that the
molecules in these crystals must have moved closer together, compared
with their corresponding positions in the wild-type and S175A mutant
crystals. These rearrangements of the molecules may have been caused by
a combination of the effects of cross-linking and conformational
changes induced by the substrate binding.
Data Collection and Processing--
Diffraction data for the
S175A mutant and all of the complex crystals were collected at 20 °C
with a Rigaku R-AXIS IIC imaging plate detector system using graphite
or double focusing mirror-monochromated CuK
radiation, produced by a
Rigaku RU-300 rotating anode x-ray generator operated at 40 kV and 100 mA. The data collection for each of the five kinds of crystals was
performed with one crystal sealed in a glass capillary. The
crystal-to-detector distance was set to 80.0 mm. Each frame of 2.6°
crystal oscillation data was collected for 10 min. Data processing was
accomplished at 2.2-1.8-Å resolution with the R-AXIS IIC data
processing software package. All of the frames of diffraction data were
merged for every data set and scaled together. Data collection and
processing are summarized in Table II.
Structure Determination and Refinement--
First of all, the
subunit structure of the S175A mutant was analyzed using the structure
of the wild-type subunit containing Tyr-3-Ile-222, or 220 out of 232 residues, which has previously been reported at 2.5-Å resolution (3)
and further refined at 2.0-Å resolution to an R-factor of
19.1% with the program X-PLOR (20). The mutant structure refined with
X-PLOR was virtually identical to the wild-type structure, with an
average r.m.s. deviation of 0.2 Å between the corresponding
C
atoms of the two structures. Therefore, structural
comparisons between the S175A mutant and its complexes allowed us to
study the reaction mechanism of the enzyme on the basis of molecular structures.
The structure determination of all the complexes except that with MCA
required some modification in position and orientation of the starting
model, because the complex crystals lacked isomorphism with the S175A
mutant crystal. Therefore, the model of the S175A subunit was first
positioned in the asymmetric unit of the CBT-complex crystal by
rigid-body refinement in X-PLOR at 8.0-3.5-Å resolution; this
required translations of
0.89, +0.01, and
0.05 Å in the x, y, and z directions, respectively,
and rotations of
0.73°,
1.30° and
0.11° in the Eulerian
angles, respectively. Each subunit of the CMB and CMV complex molecules
was located by means of a rigid-body refinement using the CBT complex
as a starting model; for the CMB (or CMV) complex, the translations
were
0.14 (
0.24),
0.02 (+0.01), and
0.05 (
0.04) Å,
respectively, in the x, y, and z
directions, and the rotations were
0.11° (
0.22°), +0.43° (+0.45°), and +0.12° (+0.17°) in the Eulerian angles,
respectively. After the subunit for each complex was located in its
asymmetric unit, the regions that had changed their conformations were
checked for each complex using the 2Fo
Fc and Fo
Fc difference electron density maps. These maps
showed that the region of Asp-10-Ser-20 in the MCA complex, and the
regions of Asp-10-Ser-20, Tyr-91-Asp-102, and Leu-117-Arg-135 in the
other three complexes had moved away from their initial positions.
Therefore, the conformations of these regions were manually modified on
a computer workstation IRIS INDIGO-Elan with the program TURBO-FRODO (21) based on the 2Fo
Fc and
Fo
Fc omit maps. The
structures were refined with the simulated annealing protocol in
X-PLOR. The refinement of each structure was initiated at 2.5-Å
resolution using the modified coordinates and the individual
temperature factors of the wild-type structure. In the initial stage of
refinement, the structures of the protein regions were refined in all
of the complexes. Some residues without any significant peaks
corresponding to their side chains were set to Ala in the first several
cycles until their peaks appeared. During the course of the refinement,
water molecules were rebuilt on the basis of the 2Fo
Fc and Fo
Fc maps. In the final stage of refinement, a model
of substrate moiety was added to the protein structure of each complex
based on the 2Fo
Fc omit map. In the map, the electron density corresponding to the substrate moiety
was connected to that of the Asp-10 side chain in each complex, as
shown in Fig. 1. The density map clearly showed that the substrate
moiety has no chlorine atom and has a D-configuration at
the asymmetric C2 atom. These findings indicated that in
each crystal, the complex turned to an ester intermediate, where the C2 atom of the unchlorinated substrate is covalently bonded
to the carboxyl oxygen atom of the Asp-10 side chain and has a
D-configuration in all but the MCA intermediate. Each of
the ester intermediate structures was further refined to convergence.
The refinement statistics are summarized in Table
III.
 |
RESULTS AND DISCUSSION |
Overall Structures of Ester Intermediates--
The structure of
each ester intermediate contains the polypeptide chain of amino acid
residues 3-222, the substrate moiety, and 66, 51, 40, or 34 water
molecules in the MCA, CBT, CMB, or CMV intermediates, respectively. The
overall structure of the polypeptide chain in each intermediate is
similar to those of the wild type and the S175A mutant, although there
are significant structural differences in the Asp-10-Ser-20,
Tyr-91-Asp-102, and Leu-117-Arg-135 regions. The conformational
change in Asp-10-Ser-20 seems to be caused by an esterification with
the substrate-derived moiety. The high quality of the appropriate
omit-maps of all the intermediate structures allowed us to precisely
locate the alkanoic acid moiety covalently bound to the carboxyl oxygen
O
2 of Asp-10 in each intermediate crystal, as shown in
Fig. 1. The CBT-, CMB-, and CMV-derived
carboxyalkyl groups have almost identical conformations. In the case of
the CMB and CMV intermediates, the L-enantiomers of CMB and
CMV were used in preparing the soaking solutions. Consequently, only
the corresponding pro-D-2-hydroxyacids were observed in
their electron density maps. In the case of the CBT intermediate, the
racemate of DL-2-chloro-n-butyrate was used in
the soaking solution, but the electron density map of the CBT intermediate unambiguously showed the conformation of
pro-D-2-hydroxybutyrate. This suggests that the enzyme
selectively reacts with the L-enantiomer of 2-haloacid and
that the C2 atom of the substrate has a completely inverted
configuration in the ester intermediates. The structures around the
active site in the MCA, CBT, CMB, and CMV intermediates are shown in
Fig. 2.

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Fig. 1.
Stereo drawing of the 2Fo Fc omit-map for the active site of the CMV
intermediate. The map contoured at the level shows the
esterified side chain of Asp-10 and the new water molecule labeled
WatN. The notation of the substrate moiety is designated on
the figure.
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Fig. 2.
Stereoscopic superpositions in the active
site structure among the wild type (black) and the MCA
(red) and CBT (blue) intermediates
(A), and among the wild type (black) and the
CMB (red) and CMV (blue) intermediates
(B). The water molecules of Wat-501 and the new water
molecule are shown by small balls labeled Wat1
and WatN, respectively. The figure was drawn using the
program MOLSCRIPT (22).
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In the MCA intermediate, the carboxymethyl group of the substrate
is covalently bonded to O
2 of Asp-10, and unesterified
O
1 of Asp-10 is hydrogen-bonded with N
of
Lys-151. In contrast, in the CBT, CMB, and CMV intermediates, O
2 of Asp-10 is esterified with the substrate moiety in
the O
1 position in the MCA intermediate, and
unesterified O
1 is hydrogen-bonded with the hydroxyl
oxygen of Thr-14, which is located opposite Lys-151. The CBT-, CMB-,
and CMV-derived moieties displace most of the water molecules observed
in the active site of the wild type, while the MCA-derived moiety
co-exists with several water molecules, probably because it is smaller
than the substrate moieties in the other intermediates. In all of the
intermediate structures, a new water molecule is commonly observed
between the carboxyl group of Asp-10 and the side chain of Ala-175,
replacing Ser-175 in the wild type. The water molecule is
hydrogen-bonded with the main-chain amido nitrogen and
N
2 of Asn-177, O
2 of Asp-180, and the
esterified O
2 of Asp-10 in all of the intermediates, and
also to the unesterified O
1 of Asp-10 in the CBT, CMB,
and CMV intermediates. These interactions are illustrated in Fig.
3.

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Fig. 3.
Schematic diagrams of the hydrogen bonds and
hydrophobic interactions in the active site of the wild type
(A) and the MCA (B) and CMV (C)
intermediates. Hydrogen bonds and hydrophobic interactions are
depicted by dotted lines with interatomic distance (Å). The
names of the hydrogen-bonding residues are shown in boxes,
those of the water molecules in oval circles, and those of
the hydrophobic residues in shaded boxes. The water
molecules of Wat-501, Wat-503, Wat-504, Wat-505, and the new water
molecule are labeled Wat1, Wat3, Wat4,
Wat5, and WatN, respectively.
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Comparison of the Wild-type and Ester Intermediate
Structures--
Conformational differences between the four ester
intermediates and the wild-type structures have been assessed by
structural comparisons. The results for the MCA and CBT intermediates
are shown in Fig. 4. The average r.m.s.
deviations in the C
atom between the MCA, CBT, CMB, and
CMV intermediates and the wild-type structures were 0.24, 0.44, 0.33, and 0.64 Å, respectively, which lie close to expected experimental
errors. This finding indicates that the overall tertiary structure of
the wild type is well preserved in the intermediates.

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Fig. 4.
Stereo view of structural comparison of the
wild-type subunit with those of the MCA and CBT intermediates. The
C backbone of the wild-type subunit (red) is
compared with those of the MCA intermediate (green) and CBT
intermediate (blue). The CBT moiety and the Asp-10 side
chain in the active site are shown in a ball-and-stick representation.
The figure was drawn with the program MOLSCRIPT (22).
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As is shown by the plots of r.m.s. C
deviation of
intermediates from the wild type against residue number in Fig.
5, however, large and small deviations in
the intermediate structures are observed mainly in the three regions of
Asp-10-Ser-20, Tyr-91-Asp-102, and Leu-117-Arg-135, although in the
MCA intermediate, only the Asp-10-Ser-20 region shows clear movements.
All these regions tend to move toward the active site. The
Asp-10-Ser-20 region containing the short
-strand
2
(Phe-16-Asp-17) and the beginning of
1 (His-19-Ala-28) exhibits
large conformational changes in all the intermediates, including
conspicuous rotations of the phenolate ring of Tyr-12 and the
side-chain carboxyl group of Asp-10. As shown in Fig. 2, the
nucleophile Asp-10 seems to have the ability to change its side-chain
conformation depending on the size of the alkyl group in the substrate.
In the ester intermediates, the carboxyl group of Asp-10 rotates by
approximately 30° around the C
-C
bond
and by approximately 80° around the
C
-C
bond. Consequently, the orientation
of the carboxyl group in the MCA intermediate is approximately opposite
to those in the other three intermediates with respect to the
C2 atom of the substrate moiety. The C
atom
of Tyr-12 moves by 1.05, 1.51, 0.67, and 0.93 Å in the MCA, CBT, CMB,
and CMV intermediates, respectively, to get closer to the active site,
and its phenolate ring rotates by approximately 30° around its
C
-C
bond in each intermediate. In the
region of Tyr-91-Asp-102, which contains
3 (Ala-95-Pro-96) and the
beginning of
5 (Val-100-Arg-109), Leu-94 shows the greatest
C
movement of 1.20, 0.51, and 0.54 Å in the CBT, CMB,
and CMV intermediates, respectively. In the Leu-117-Arg-135 region,
which contains the end of
4 (Lys-113-Ser-118) and the whole
6
(Pro-122-His-131), the Ser-118-Ala-127 region shows large changes
with C
-averaged shifts of 1.38, 0.64, and 0.90 Å in the
CBT, CMB, and CMV intermediates, respectively. The structural changes
described above in the Asp-10-Ser-20 region are caused by the
nucleophilic attack of Asp-10 on the substrate, while those in the
Tyr-91-Asp-102 and Leu-117-Arg-135 regions can be attributed to the
bulkiness of the alkyl group in the CBT, CMB, and CMV substrates. The
structural differences between the ester intermediates and the wild
type indicate that, in the reaction process, conformational changes favorable to the formation of intermediates occur in the active site.

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Fig. 5.
Plots of r.m.s. C deviation of
intermediates from the wild type against residue number. Plots
were drawn with the CCP4 program (23). Structural formulas of
substrates used for intermediate preparation are shown as
insets. Regions with large r.m.s. deviation are marked with
arrows and names.
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Some differences in intersubunit interactions in the molecule are
observed between the intermediates and the wild type. The intersubunit
hydrogen bond between N
2 of Asn-42 in one subunit and
O
1 of Glu-46 in the other, which is observed in the
wild-type enzyme, disappears in all the intermediates. Intersubunit
hydrophobic interactions that occur between the side chains of Trp-49
and Leu-50, Leu-50 and Pro-152, Trp-49 and Leu-53, and Leu-53 and Pro-152 in the wild-type structure generally seem to be weakened in the
ester intermediate structures.
The Binding Site of the Substrate Carboxyl Group--
The
dehalogenated moieties of the substrates in the four intermediates
appear to have similar positions and orientations to one another. In
particular, the carboxyl group of the substrate moiety in each
structure occupies a very similar position, although some differences
in the orientation of the alkyl group in the substrate-derived moieties
are observed between the CBT, CMB, and CMV intermediates because of
differences in the branching position. The relationships between the
carboxylates of the substrate-derived moieties and their neighboring
polypeptide residues are shown in Figs. 2 and 3. In the structures of
the CBT, CMB, and CMV intermediates, the carboxyl oxygens of each
substrate moiety are hydrogen-bonded with the Ser-118 hydroxyl and the
main-chain amido nitrogens of Leu-11, Tyr-12, and Asn-119. The hydrogen
bond between the carboxyl oxygen and the main-chain amido nitrogen of
Asn-119 is not observed in the MCA intermediate. These structures
clearly show that Ser-118 serves as the main residue for stabilizing
the substrate carboxyl moiety in the reaction intermediate. The
position of the hydroxyl oxygen atom of Ser-118 in the wild type is
very close to those in the intermediates. Therefore, it is reasonable
to expect that Ser-118 should be a residue essential for binding the
carboxyl group of the substrate through the hydrogen bonds.
The Hydrophobic Pocket for the Substrate Alkyl Group--
The
alkyl groups derived from CBT, CMB, and CMV in the ester intermediates
are situated in a unique binding site of the present enzyme. The
hydrophobic side chains of Tyr-12, Leu-45, Phe-60, and Trp-179 are
located in the vicinity of the binding site, as shown in Fig. 2. The
four residues obviously form the hydrophobic pocket for the alkyl
group, along with the Gln-44, Lys-151, and Asn-177 side chains. This
hydrophobic pocket lies at the bottom of the cleft between the
core-domain and subdomain, and some of the constituent residues are
incorporated into the hydrophobic cluster, which contributes to the
formation of the dimer molecule, as described previously (3). The seven
residues that form the hydrophobic pocket are conserved in most
L-DEXs thus far sequenced. We have shown by site-directed
mutagenesis studies that Lys-151 and Asn-177 are essential for the
enzyme activity (7). The mutagenesis studies also showed that the W179F
mutant is only 10% as active as the wild-type enzyme (7), and that
mutants for Tyr-12 show somewhat higher activities of 11, 11, 40, and 16% in Y12A, Y12L, Y12F, and Y12W, respectively. These Tyr-12 and
Trp-179 mutations suggest that hydrophobic residues of an appropriate
size are required for the residues in these positions to make
sufficiently hydrophobic interactions with the substrate. In the
intermediate structures, the distance from the C2 atom of
the substrate moiety to the van der Waals surface at the bottom of the
hydrophobic pocket is about 6 Å (Fig.
6). This value is equivalent to the size
of the pocket, which can accommodate straight alkyl-chains of maximally
five carbons, which explains the substrate specificity of
L-DEX YL (1, 17). Based on the structure of the ester
intermediate, the possibility of a D-substrate binding to
the enzyme can easily be examined by exchanging the positions of the
hydrogen atom and the alkyl group that are attached to the
C2 atom of the substrate moiety. It turns out, however,
that no alkyl groups could be accommodated properly in the place of hydrogen due to steric hindrance by the main-chain and side-chain atoms
of Leu-11, Tyr-12 and their neighbors. Therefore, it seems reasonable
to assume that the enzyme can select only L-enantiomers of
2-haloacids as its substrates using the hydrophobic pocket. The
hydrophobic pocket of L-DEX YL may determine not only the substrate specificity of the enzyme but also its stereospecificity by
selectively accommodating the alkyl side chain of
L-2-haloacid in the inside of the pocket.

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Fig. 6.
Stereo view of interactions around the active
site, depicted as van der Waals dot surfaces. The van der Waals
surface of Arg-41 present at the entrance to the active site is shown
in green, that of Asp-10 in pale yellow, and the
others in blue.
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Proposed Catalytic Mechanism--
L-DEX YL initiates
the dehalogenation reaction by taking a L-2-haloacid into
the active site. When the substrate approaches the entrance to the
active site, the guanidino group of Arg-41 may serve as the recognition
site for the substrate carboxyl group. Arg-41 is the only basic residue
present at the entrance of the active site (Fig. 6). This residue is
entirely conserved in all L-DEXs and is essential for the
enzyme activity, as has been evidenced by site-directed mutagenesis
studies (7).
The enzyme forms a Michaelis complex in the active site with the
substrate. When the complex is formed, the water molecule Wat-504 is
displaced or removed by the substrate carboxyl gruop. In the complex,
the position of Wat-504 is occupied by the carboxyl group of the
substrate, as is the case with all the intermediates. The carboxyl
group of the substrate moiety is present in a position common to all
the ester intermediates. The situation of the substrate carboxyl group
in the intermediates resembles that of the water molecule Wat-504 in
the substrate-free form of the wild-type enzyme. The water molecule
Wat-501, which is hydrogen-bonded to both O
1 and
O
2 of Asp-10 in the substrate-free enzyme, is removed
upon the formation of the ester intermediate. The O
2
atom of Asp-10 probably serves as a nucleophile attacking the C2 atom of the substrate, because in the wild-type enzyme,
the O
1 atom of Asp-10 is stabilized by hydrogen bonding
with O
of Ser-175 as well as Wat-501. Whichever oxygen
atom of Asp-10 takes part in the reaction, the liberation of the
nucleophilic oxygen atom from Wat-501 and Wat-504 upon the binding of
the substrate is probably the crucial step in the reaction.
The dehalogenation of L-2-haloacid catalyzed by
L-DEX YL probably proceeds through an SN2
mechanism; the carboxyl group of Asp-10 is assumed to approach the
C2 atom of the substrate from the opposite side to the
halogen atom. In the transition state of the reaction, the groups other
than the leaving group that are attached to the C2 atom
become planar with the atom. The nucleophile O
2 of
Asp-10 attacks the C2 atom from a direction perpendicular to the plane, while the leaving halide anion is abstracted concertedly by a residue located on the opposite side of the plane.
One of the issues to be solved is what residue serves as the acceptor
of the halide ion released from the substrate. We previously proposed
that Tyr-12 in the vicinity of Asp-10 could be an acceptor for the
halide ion based on the structure of the substrate-free enzyme (3).
However, further site-directed mutagenesis studies have shown that the
Y12A and Y12L mutant enzymes have more than 10% of the activity of the
wild-type enzyme described above. Moreover, the phenyl ring of Tyr-12
is not in a proper position with respect to the C2 atom of
the substrate moiety in the ester intermediate, although the residue is
in the vicinity of Asp-10. These findings do not support our earlier
proposal. A candidate residue for the halide ion acceptor is Arg-41;
Arg-41 is the only functional residue that appears to be in a suitable
position in all ester intermediates (Fig. 6). Arg-41 is located in a
proper position with respect to the C2 atom of the
substrate moiety. It turns out that the O
2 atom of
Asp-10, the C2 atom of the substrate, and the
N
1 atom of Arg-41 line up approximately on a straight
line. The angles between the Asp-10 oxygen O
2 bound to
the substrate C2 atom and N
1 of Arg-41 with
respect to the C2 atom are approximately 123°, 149°,
156°, and 150° in the MCA, CBT, CMB, and CMV intermediates, respectively. The distance between N
1 of Arg-41 and the
C2 atom of the substrate is also short enough for Arg-41 to
interact with the chlorine atom: 8.42, 5.45, 5.60, and 5.68 Å in the
MCA, CBT, CMB, and CMV intermediates, respectively. These findings seem
to suggest that the guanidino group of Arg-41 serves as the halogen
abstraction site. The relatively smaller angle and longer distance
observed in the MCA intermediate is probably attributable to the lack
of an alkyl side chain in MCA, which probably permits the presence of
water molecules and therefore affects the position of the guanidino
group of Arg-41.
The possibility that Arg-41 functions as the halogen abstraction
residue was examined by means of a hypothetical model compound mimicking the transition-state structure in the SN2
reaction of L-DEX YL. The model compound contained the
C2 atom, a hydrogen atom, a carboxyl group and an alkyl
group that are attached to the C2 atom on a plane, and also
a chlorine atom bound to the C2 atom in a direction
perpendicular to the plane. The chlorine of the hypothetical compound
was oriented toward the guanidino group of Arg-41. The carboxyl group
of Asp-10 was shifted to a new position so that the
O
2-C
bond of Asp-10 could be oriented
toward the C2 atom of the compound and perpendicularly to
the plane of the compound. The other conditions in the transition-state
model were fixed to those observed in the ester intermediates.
Consequently, as shown in Fig. 7,
C
and O
2 of Asp-10, and the chlorine and
C2 atom of the hypothetical compound lie linearly in the
transition state. The model shown in Fig. 7 is a reasonable
transition-state model of the proposed SN2-type reaction,
and it allows us to expect that the guanidino group of Arg-41 probably
abstracts the halide ion from the L-2-haloacid. As the
C2-O
2 bond is formed between the substrate
and Asp-10 with the concomitant abstraction of the halide anion in the
transition state, the orientation of the other three bonds to the
C2 atom are inverted with respect to the
C2-O
2 bond, and so a Walden inversion is
accomplished (Fig. 8). In the formation
of the C2-O
2 bond, Asp-10 markedly changes
the conformation of its side chain as well as its main chain,
especially by rotations around the C
-C
and C
-C
bonds. Consequently, if the
substrate contains a bulky alkyl group like that of CBT, CMB, or CMV,
then the O
2-C
bond of Asp-10 probably
rotates around the C2-O
2 bond concomitantly
with the Walden inversion because of steric hindrance. In fact, the
orientation of the carbonyl group C
=O
1 in
the ester intermediates varies according to the substrates used; the
carbonyl oxygen O
1 is oriented toward N
of Lys-151 in the MCA intermediate, whereas it projects toward O
1 of Thr-14 located on the opposite side of Lys-151 in
the other intermediates. Therefore, in the case of the MCA
intermediate, residue Lys-151, which is tightly hydrogen-bonded with
the unesterified oxygen O
1 of Asp-10, probably acts as a
component residue of the oxyanion hole for stabilizing the negative
charge accumulated on the O
1 atom, resulting in a
nucleophilic attack on the C
atom of the Asp-10 by an
activated water. On the other hand, in the cases of CBT, CMB, and CMV
intermediates, Thr-14 is the main residue of oxyanion hole since its
O
1 atom is tightly hydrogen-bonded with
O
1 of Asp-10, while Lys-151 is weakly hydrogen-bonded
with the esterified oxygen O
2 of the Asp-10. In the wild
type, the hydroxyl group of Ser-175 is hydrogen-bonded with
O
1 of Asp-10, although this interaction is not observed
in the S175A mutant. Therefore, Ser-175 is likely to act as an
additional residue of the oxyanion hole.

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Fig. 7.
A postulated planar transition-state compound
existing in the active site just before the formation of the CBT
intermediate. The CBT molecule in the transition state is shown in
a ball-and-stick representation with the chlorine atom as a large
black ball. Possible hydrogen bonds are shown by dotted
lines. The figure was drawn with the program MOLSCRIPT (22).
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Fig. 8.
A proposed mechanism for the first step of
the reaction. A proposed reaction mechanism for the completion of
the Walden inversion is illustrated in the figure. A detailed
description is given in the text. There are two possible routes from
the transition state to the ester intermediate. a, the side
chain of Asp-10 remains in the conformation of the transition state in
the case of MCA; b, it rotates around the
O 2-C bond in the other cases, resulting
in a conformation markedly different from that observed with MCA.
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The water molecule newly appearing near Ala-175 and Asn-177 in each
ester intermediate is probably utilized for the hydrolysis of the ester
bond because it occupies a position near the ester bond, and is
hydrogen-bonded with the side chains of Asp-10, Asn-177, and Asp-180.
The latter two residues are essential for hydrolysis.2 We
have obtained evidence, through ion-spray mass spectrometry, showing
that a step after the ester formation such as the hydrolysis of the
ester is rate-determining in the dehalogenation of
L-2-haloacids catalyzed by L-DEX YL. Probably,
the formation of proper hydrogen bonds between the water molecule and
the side chains of Ser-175, Asn-177 and Asp-180 is essential for the
hydrolysis of the ester. Our mass spectroscopic study has suggested
that Asn-177 and Asp-180 are also involved in the hydrolysis of the
ester intermediate.2
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-aid for
Scientific Research 09660088 (to Y. H.), 08760080 (to T. K.),
and 08680683 and 09460049 (to N. E.) from the Ministry of
Education, Science, Sports and Culture of Japan, and by a research
grant from the Japanese Society for the Promotion of Science (Research
for the Future) (to N.E.).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 (codes 1ZRM and
1ZRN) have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, Upton, NY.
§
Member of the Tsukuba Advanced Research Alliance (TARA) project of
Tsukuba University, Japan. To whom correspondence should be addressed.
Tel.: 81-774-38-3251; Fax: 81-774-38-3014; E-mail: hata{at}scl.kyoto-u.ac.jp.
To whom correspondence should be addressed. Tel.:
81-774-38-3241; Fax: 81-774-38-3248; E-mail:
esaki{at}scl.kyoto-u.ac.jp.
1
The abbreviations used are: L-DEX,
L-2-haloacid dehalogenase; L-DEX YL,
L-2-haloacid dehalogenase from Pseudomonas sp.
YL; MCA, monochloroacetate; CPA, L-2-chloropropionate; CBT,
L-2-chloro-n-butyrate; CMB,
L-2-chloro-3-methyl-n-butyrate; CMV,
L-2-chloro-4-methyl-n-valerate; MCA, CBT, CMB,
and CMV intermediates, reaction intermediates between the S175A mutant
and the MCA, CBT, CMB, or CMV reagent, respectively; r.m.s., root mean
square.
2
Y.-F. Li, manuscript in preparation.
 |
REFERENCES |
-
Hasan, A. K. M.,
Takada, H.,
Koshikawa, H.,
Liu, J.-Q.,
Kurihara, T.,
Esaki, N.,
and Soda, K.
(1994)
Biosci. Biotechnol. Biochem.
58,
1599-1602
-
Hisano, T.,
Hata, Y.,
Fujii, T.,
Liu, J.-Q.,
Kurihara, T.,
Esaki, N.,
and Soda, K.
(1996)
Proteins Struct. Funct. Genet.
24,
520-522[CrossRef][Medline]
[Order article via Infotrieve]
-
Hisano, T.,
Hata, Y.,
Fujii, T.,
Liu, J.-Q.,
Kurihara, T.,
Esaki, N.,
and Soda, K.
(1996)
J. Biol. Chem.
271,
20322-20330[Abstract/Free Full Text]
-
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.,
Verschueren, K. H. G.,
and Goldman, A.
(1992)
Protein Eng.
5,
197-211[Abstract]
-
Liu, J.-Q.,
Kurihara, T.,
Miyagi, M.,
Esaki, N.,
and Soda, K.
(1995)
J. Biol. Chem.
270,
18309-18312[Abstract/Free Full Text]
-
Liu, J.-Q.,
Kurihara, T.,
Miyagi, M.,
Tsunasawa, S.,
Nishihara, M.,
Esaki, N.,
and Soda, K.
(1997)
J. Biol. Chem.
272,
3363-3368[Abstract/Free Full Text]
-
Kurihara, T.,
Liu, J.-Q.,
Nardi-Dei, V.,
Koshikawa, H.,
Esaki, N.,
and Soda, K.
(1995)
J. Biochem. (Tokyo)
117,
1317-1322[Abstract]
-
van der Ploeg, J.,
van Hall, G.,
and Janssen, D. B.
(1991)
J. Bacteriol.
173,
7925-7933[Medline]
[Order article via Infotrieve]
-
Verschueren, K. H. G.,
Franken, S. M.,
Rozeboom, H. J.,
Kalk, K. H.,
and Dijkstra, B. W.
(1993)
J. Mol. Biol.
232,
856-872[CrossRef][Medline]
[Order article via Infotrieve]
-
Verschueren, K. H. G.,
Seljée, F.,
Rozeboom, H. J.,
Kalk, K. H.,
and Dijkstra, B. W.
(1993)
Nature
363,
693-698[CrossRef][Medline]
[Order article via Infotrieve]
-
Lacourciere, G. M.,
and Armstrong, R. N.
(1993)
J. Am. Chem. Soc.
115,
10466-10467
-
Yang, G.,
Liang, D.-H.,
and Dunaway-Mariano, D.
(1994)
Biochemistry
33,
8527-8531[Medline]
[Order article via Infotrieve]
-
Klages, U.,
Krauss, S.,
and Lingens, F.
(1983)
Hoppe-Seyler's Z. Physiol. Chem.
364,
529-535[Medline]
[Order article via Infotrieve]
-
Benning, M. W.,
Tayler, K. L.,
Liu, R.-Q.,
Yang, G.,
Xiang, H.,
Wesenberg, G.,
Dunaway-Mariano, D.,
and Holden, H. M.
(1996)
Biochemistry
35,
8103-8109[CrossRef][Medline]
[Order article via Infotrieve]
-
Crooks, G. P.,
Xu, L.,
Barkley, R. M.,
and Copley, S. D.
(1995)
J. Am. Chem. Soc.
117,
10791-10798
-
Liu, J.-Q.,
Kurihara, T.,
Esaki, N,
and Soda, K.
(1994)
J. Biochem. (Tokyo)
116,
248-249[Abstract]
-
Liu, J.-Q.,
Kurihara, T.,
Hasan, A. K. M. Q.,
Nardi-Dei, V.,
Koshikawa, H.,
Esaki, N.,
and Soda, K.
(1994)
Appl. Environ. Microbiol.
60,
2389-2393[Abstract]
-
Kunkel, T. A.,
Roberts, J. D.,
and Zakour, R. A.
(1987)
Methods Enzymol.
154,
367-382[Medline]
[Order article via Infotrieve]
-
Iwasaki, I.,
Utsumi, S.,
Hagino, K.,
and Ozawa, T.
(1956)
Bull. Chem. Soc. Jpn.
29,
860-864
-
Brünger, A. T.
(1992)
X-PLOR Manual, Version 3.1, Yale University Press, New Haven, CT
-
Jones, T. A.
(1985)
Methods Enzymol.
115,
157-171[Medline]
[Order article via Infotrieve]
-
Kraulis, P.
(1991)
J. Appl. Crystallogr.
24,
946-950[CrossRef]
-
Collaborative Computational Project Number 4.
(1994)
Acta Crystallogr. Sec. D
50,
760-763
[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.