1 leukotriene a4 hydrolase: identification of a common carboxylate
TRANSCRIPT
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Leukotriene A4 hydrolase: identification of a common carboxylate recognition site
for the epoxide hydrolase and aminopeptidase substrates
Peter C. Rudberg1*, Fredrik Tholander1*, Martina Andberg1, Marjolein M.G.M. Thunnissen2
and Jesper Z. Haeggström1§
1 Department of Medical Biochemistry and Biophysics, Division of Chemistry II,
Karolinska Institutet, S-171 77 Stockholm, Sweden.
2 Department of Molecular Biophysics, Kemicentrum, Lund University, S-221 00 Lund,
Sweden.
* These authors contributed equally to the present study
§ To whom correspondence should be addressed:
Tel +46-8-728 76 12, Fax +46-8-736 04 39, E-Mail: [email protected]
Running title: A dual carboxylate recognition site in LTA4 hydrolase
JBC Papers in Press. Published on April 12, 2004 as Manuscript M401031200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Abstract
Leukotriene (LT) A4 hydrolase is a bifunctional zinc metalloenzyme, which converts LTA4
into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent
aminopeptidase activity. In the X-ray crystal structure of LTA4 hydrolase, Arg563 and
Lys565 are found at the entrance of the active center. Here we report that replacement of
Arg563, but not Lys565, leads to complete abrogation of the epoxide hydrolase activity.
However, mutations of Arg563 do not seem to affect substrate binding strength, since values
of Ki for LTA4 are almost identical for wild type and [R563K]LTA4 hydrolase. These results
are supported by the 2.3 Å crystal structure of [R563A]LTA4 hydrolase, which does not
reveal structural changes that can explain the complete loss of enzyme function. For the
aminopeptidase reaction, mutations of Arg563 reduce the catalytic activity (Vmax=0.3-20%),
whereas mutations of Lys565 have limited effect on catalysis (Vmax =58-108%). However, in
[K565A]- and [K565M]LTA4 hydrolase, i.e. mutants lacking a positive charge, values of the
Michaelis constant for alanine-p-nitroanilide increase significantly (Km=480-640%).
Together, our data indicate that Arg563 plays an unexpected, critical role in the epoxide
hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect
substrate alignment along the catalytic elements of the active site. In the aminopeptidase
reaction, Arg563 and Lys565 seem to cooperate to provide sufficient binding strength and
productive alignment of the substrate. In conclusion, Arg563 and Lys565 possess distinct
roles as carboxylate recognition sites for two chemically different substrates, each of which is
turned over in separate enzymatic reactions catalyzed by LTA4 hydrolase.
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Introduction
The leukotrienes (LTs) are a class of structurally related lipid mediators involved in the
development and maintenance of inflammatory and allergic reactions (1,2). In the
biosynthesis of LTs, 5-lipoxygenase converts arachidonic acid into the unstable epoxide
LTA4. This intermediate may in turn be conjugated with glutathione to form the spasmogenic
LTC4, or hydrolyzed into the proinflammatory lipid mediator LTB4, in a reaction catalyzed by
LTA4 hydrolase (LTA4H). Leukotriene B4 is a classical chemoattractant of human neutrophils
and triggers adherence and aggregation of leukocytes to vascular endothelium at only
nanomolar concentrations (3). In addition, LTB4 modulates immune responses (4,5) and
recent studies show that this lipid mediator is involved in early effector CD4+ and CD8+ T cell
recruitment to sites of inflammation (6-8). Moreover, LTB4 participates in the host-defense
against infections (9,10) and is a key mediator of PAF-induced lethal shock (11-13). These
effects are signaled via specific, G-protein coupled receptors for LTB4 (BLT1 and BLT2)
(14,15). The relative contributions of these two receptors to signaling and bioactivity are
presently not clear.
LTA4H (EC 3.3.2.6) is a bifunctional zinc metalloenzyme, exhibiting an anion-
dependant aminopeptidase activity in addition to its epoxide hydrolase activity, i.e. the
hydrolysis of the unstable allylic epoxide LTA4 into LTB4, for a review see (16). Unlike the
aminopeptidase activity, the epoxide hydrolase activity is restrained by suicide inactivation
which involves binding of LTA4 to Tyr378, which is located within a 21-residue active-site
peptide denoted K21 (17,18). The epoxide hydrolase reaction of LTA4H is also unique in the
sense that the stereoselective introduction of water to the carbon backbone of LTA4 occurs
several methylene units away from the epoxide moiety, presumably proceeding via a
carbocation intermediate (19). Furthermore, Asp375 was shown to assist in the introduction of
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the 12R hydroxyl group of LTB4 (20), a key component for the biologic activity of LTB4 (21-
23).
Certain arginyl di- and tripeptides as well as p-nitroanilide derivatives of Ala and Arg
are very efficient substrates for the aminopeptidase activity (24). Although it has never been
proven, it is generally believed that the peptidase activity is involved in the processing of
bioactive peptides related to inflammation and host-defense. Several lines of evidence
indicate that the aminopeptidase activity follows a zinc-assisted general base mechanism with
Glu296 and Tyr383 acting as the general base and proton donor, respectively. Moreover, in
recent studies, Glu271 was identified as the recognition site for the N-terminal amino group
of the peptidase substrate (25). Recently, we solved the crystal structure of LTA4H in
complex with the aminopeptidase inhibitor bestatin at 1.95 Å resolution, which has given
important clues to the molecular mechanisms of the two enzyme reactions (26).
Earlier work with chemical modification already indicated the presence of essential Arg
residues near or at the active center of LTA4H (27). From the X-ray crystal structure of
LTA4H, a positively charged site was identified in a cavity formed between the N-terminal,
the central catalytic, and the C-terminal domain (Fig. 1). This site, composed of Arg563 and
Lys565, is located in a wide portion of the cavity, near its entrance, and in the vicinity of the
carboxyl group of bestatin. These two residues are positioned in the first turn of an α-helix
pointing towards the active site. While Arg563 is buried, Lys565 is fully exposed to solvent.
Also, when LTA4 was modeled into the active site, electrostatic interactions seemed
possible between the C1 carboxylate of LTA4 and the positively charged side groups of
Arg563 and/or Lys565 (Fig. 1). Furthermore, in the crystal structure of LTA4H in complex
with a specific hydroxamic acid inhibitor that is a structural mimic of LTA4, a direct
interaction between the carboxylic moiety of this inhibitor and Arg563 is seen (28). In this
report we used site-directed mutagenesis combined with X-ray crystallography and inhibition
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studies to analyze the functional role of Arg563 and Lys565 as putative carboxylate
recognition sites in LTA4H. We show that Arg563 is required for the epoxide hydrolase
activity, presumably in an indirect manner to achieve correct alignment of LTA4 along the
catalytic elements of the active site. As a result, Arg563 is a key residue during LTB4
formation. For the aminopeptidase reaction, we propose that Lys565 assists Arg563 for
optimal substrate binding. Thus, we have identified a common carboxylate recognition site for
lipid and peptide substrates in the active center of LTA4H.
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Experimental procedures
Materials
Oligonucleotides were synthesized by Cybergene, Stockholm, Sweden. LTA4 methyl ester
was synthesized as described (29) or purchased from BIOMOL Res. Lab, Plymouth Meeting,
PA, USA. LTA4 methyl ester was saponified in tetrahydrofuran with 1M LiOH (6% v/v) for
48 h at 4ºC. Alanine-p-nitroanilide, isopropyl-β-D-thiogalactopyranoside,
phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and streptomycin sulphate were
purchased from Sigma, Sweden. Nickel nitrilo-triacetic-acid resin was from QIAGEN.
Mutagenesis of human LTA4H cDNA
Site-directed mutagenesis was carried out by PCR on the recombinant plasmid pT3MB5 for
expression of (His)6-tagged LTA4H in E. coli. This plasmid is a variant of pT3MB4 (18) in
which an Nsi I restriction site has been eliminated by a single nucleotide exchange. Briefly,
site-directed mutagenesis involves two consecutive steps of PCR comprising four different
primers, according to the megaprimer method (30). Polymerase chain reactions were carried
out in a total volume of 50 µl, using 1x Pfu Polymerase Buffer, 125-150 ng each of primers A
and B, 1 U Pfu polymerase, 10 nmol each of dNTPs and 100 ng template. In the second
reaction, 15-20 µl of the first PCR mix was used to supply the reaction with the megaprimer,
which was used together with a primer C (125-150 ng). The amplification program included
an initial round of denaturation at 94°C (60 s), annealing at 55-66°C (60 s) and elongation at
72°C (90 s), followed by 30 cycles of denaturation (45 s), annealing (30 s) and elongation (60
s) on a PE GeneAmp PCR System 2400.
For generation of [E560Q]-, [R563K]-, [R563A]-, [R563M]-, [K565R]-, [K565A]-,
[K565M]- and [R563K/K565R]LTA4H, a Bfr I and a Nsi I site were used. DNA fragments
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were cleaved with Bfr I/Nsi I and purified by agarose gel electrophoresis (1.5 %) followed by
extraction (QIAEX II Gel Extraction Kit). Mutated fragments were ligated into pT3MB5 (T4
DNA Ligase Protocol), opened with the same restriction enzymes. Competent E. coli cells
(JM101) were transformed with mutated recombinant plasmid and grown in LB medium
containing ampicillin (100 µg/ml). Stock cultures were kept at -70°C in a 1:1 mixture of
culture medium and 40 % (v/v) glycerol/0.75 % (w/v) NaCl, respectively. Recombinant
plasmids were purified using Wizard Minipreps Plus and the entire mutated inserts were all
sequenced using Dyenamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia
Biotech) to confirm that no nucleotide alterations had occurred in addition to the desired
mutation.
Protein expression and purification
Mutated enzymes were expressed as N-terminal (His)6-tag fusion proteins in E. coli (JM101)
cells grown at 37°C in M9 medium (50 mM Na2HPO4, 22 mM KH2PO4, 20 mM NH4Cl, 8.5
mM NaCl), pH 7.4, containing 0.4 % glucose (w/v), 0.2 % (w/v) casamino acids, 2 mM
MgSO4 and 0.1 mM CaCl2. At OD620 ≈ 0.2, isopropyl-β-D-thiogalactopyranoside was added
to a final concentration of 500 µM. Cells were harvested at OD620 ≈ 1.8, pelleted at 1000 x g
and resuspended in 30 ml homogenization buffer (50 mM Tris-HCl, pH 8.0, containing
soybean trypsin inhibitor) supplemented with phenylmethylsulfonyl fluoride (1 mM). Nucleic
acids were removed by streptomycin sulphate precipitation. After centrifugation (10 000 x g
for 15 min), the supernatant was filtered (0.22 µm) and applied to a nickel nitrilo-triacetic-
acid resin. The column was washed with 1 bed volume of 50 mM Tris-HCl, pH 8.0, 50 mM
sodium phosphate buffer, pH 6.8, containing 0.5 M NaCl, and 50 mM Tris-HCl, pH 8.0, with
each solution supplemented with 10 mM imidazole. The His-tagged protein was eluted with
1.4 bed volumes of 50 mM Tris-HCl, pH 8.0, containing 100 mM imidazole. The purity of the
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final preparation was assessed by SDS-PAGE on a Pharmacia Phast system, using 10-15 %
gradient gels, subsequently stained with Coomassie brilliant blue. Protein concentrations were
determined by the Bradford method using a MCC/340 96-well multiscan spectrophotometer
and BSA as standard (31).
Aminopeptidase activity assays
The aminopeptidase activity, expressed as Vmax, was determined in a spectrophotometric assay
at 405 nm, using a MCC/340 multiscan spectrophotometer, essentially as described (32).
Briefly, the enzyme (1-20 µg) was incubated at room temperature in 96-well microtiter plates
with alanine-p-nitroanilide as substrate in 50 mM Tris-HCl, pH 8.0, containing 100 mM KCl.
The absorbance at 405 nm was measured at 10 min intervals. Different concentrations of
substrate (0.125, 0.25, 0.5, 1, 2, 4, 8 mM) were used for kinetic determinations. Spontaneous
hydrolysis of the substrate was corrected for by subtracting the absorbance of blank
incubations without enzyme. The Michaelis constant (Km) and Vmax were determined by
plotting the calculated velocity as a function of substrate concentration, whereby a non-linear
regression analysis was performed.
Epoxide hydrolase activity assays and reverse-phase HPLC
The epoxide hydrolase activity, expressed as Vmax, was determined from incubations of
enzyme (1-20 µg) in 100 µl 10 mM Tris-HCl, pH 8.0, with LTA4 (2.5-125 µM) for 30 s on
ice. Reactions were quenched with 200 µl of MeOH, followed by the addition of 0.4 nmol of
prostaglandin B1 (PGB1) or PGB2 as internal standard. Samples were acidified with 5 µl of
acetic acid (10 %), and metabolites were extracted on solid phase Chromabond C18 columns.
Metabolites of LTA4 were separated by isocratic reverse-phase HPLC on a Waters Nova-Pak
C18 column eluted with a mixture of methanol/acetonitrile/water/acetic acid (30:30:40:0.01
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by vol.) at a flow rate of 1.2 ml/min. The UV detector was set at 270 nm and metabolites were
quantified using Chromatography Station for Windows version 1.7 computer software.
Calculations were based on peak area measurements and the known extinction coefficients for
the internal standards PGB1 and PGB2 (30 000 M-1 x cm-1) as well as LTB4 (50 000 M-1 x cm-
1). The Michaelis constant (Km) and Vmax were determined by plotting the calculated velocity
as a function of substrate concentration, whereby a non-linear regression analysis was
performed.
Inhibition assays
Prior to performing aminopeptidase activity assays, wild type-, [R563K]- and
[R563M]LTA4H were pre-incubated with a hydroxamic acid inhibitor, 0.01 – 0.3 µM or
LTA4, 1-10 µM. Aminopeptidase assays were then performed as described.
Crystallization
Plate-like crystals of [R563A]LTA4H were obtained by liquid-liquid diffusion in capillaries,
as previously described (26). Briefly, 5 µl of precipitation solution (28 % (v/v) PEG8000, 0.1
mM Na acetate, 0.1 mM imidazole buffer, pH 6.8, and 5 mM YbCl3), was injected into the
bottom of a melting point capillary and an equal volume of [R563A]LTA4H (5 mg/ml) in 10
mM Tris-HCl, pH 8, containing 1 mM bestatin, was layered on top.
Data collection and structure determination
For data collection, crystals were soaked in 15% (w/v) PEG8000, 50 mM sodium-acetate, 50
mM imidazole buffer, pH 6.8, 2.5 mM YbCl3 and 25% (v/v) glycerol. Data was collected at
the beamline I711 of Max-Lab, Lund, Sweden. A complete set of data was collected from a
single crystal. Statistics on data collection and quality are given in Table I. The crystals
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belonged to space-group P212121 with cell dimensions a=78 b=86.85 c=98.95, α=β=γ=90° at
100 K. Processing, scaling and merging of data were carried out using the program Mosflm
(33) and programs from CCP4 (34). The [E271Q]LTA4H mutant structure (25) was used as
the starting point for refinement. All refinement was done using the CNS package (35).
Manual model building as well as interpretation of electron density maps was performed
using the program XtalView (36). During the refinement, 614 water molecules, one Zn2+ ion,
one imidazole molecule, one acetate molecule, and one Yb3+ ion were identified. The final R-
factor was 18.8 % and the Rfree factor was 23.9 %. 3.3% of total reflections were set aside to
calculate the Rfree. Most of the model of [R563A]LTA4H is in good density except for the
(His)6-tag and the first four N-terminal residues. In the model, 99.4 % of the residues are
confined to the most favorable or additionally allowed regions and 0.6 % in the generously
allowed regions of the Ramachandran plot. R.m.s. deviations for bond lengths and angles
were 0.0079 Å and 1.44°.
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Results
Mutagenetic replacements, expression and purification of recombinant proteins
To detail the function of the Arg/Lys site in LTA4H we exchanged Arg563 for a Lys, Ala or
Met, and Lys565 for an Arg, Ala or Met, generating the mutants [R563K]-, [R563A]-,
[R563M]-, [K565R]-, [K565A]- and [K565M]LTA4H. We also reversed the positions of
Arg563 and Lys565, generating the double cross-over mutant [R563K/K565R]LTA4H. As
control, we exchanged Glu560 for a Gln, generating [E560Q]LTA4H. This control residue
was selected on the basis of proximity to the active site and predicted non-catalytic nature.
The resulting 8 mutants were all expressed as (His)6-tagged fusion proteins in E. coli, to allow
rapid purification on nickel-NTA resins. The level of expression was similar for wild type
enzyme and all mutants, with a final yield of 2.4 to 6.5 mg purified protein per liter cell
culture.
Catalytic properties of mutants of Arg563
Mutagenetic replacements of Arg563 completely eliminated the epoxide hydrolase activity of
LTA4H (Table II). Thus, neither [R563K]-, [R563A]-, nor [R563M]LTA4H converted LTA4
into detectable amounts of LTB4. Although these mutations also strongly reduced the
aminopeptidase activity, the effects were not quite as detrimental as for the epoxide hydrolase
reaction. Thus, in [R563K]LTA4H, Vmax and Km reached 6.4 and 58%, respectively, of wild
type enzyme, while in [R563A]LTA4H, the aminopeptidase activity was almost completely
abolished, with a Vmax and Km for Ala-p-NA of 0.3 and 6.2 %, respectively. On the other
hand, [R563M]LTA4H exhibited a Vmax and Km of 20 and 179%, respectively, of wild type
enzyme (Table II). Accordingly, the specificity constants of both [R563K]- and
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[R563M]LTA4H were reduced to only 11% of the wild type enzyme, whereas the specificity
constant for [R563A]LTA4H only reached 5%.
Catalytic properties of mutants of Lys565
Mutations of Lys565 had variable, but no dramatic effects on the epoxide hydrolase activity.
[K565R]LTA4H exhibited a Vmax and Km of 95 and 104%, respectively, of wild type enzyme,
whereas for [K565A]LTA4H, Vmax and Km for LTA4 were 31 and 14%, respectively, and for
[K565M]LTA4H these values mounted to 32 and 33%, respectively, of wild type enzyme
(Table II). In addition, the specificity constants for the more conservative mutant
[K565R]LTA4H and for [K565M]LTA4H remained unchanged (95 and 92%, respectively)
and even increased for [K565A]LTA4H (218%), indicating that the substrate binding and
catalytic residues are not drastically altered in these mutants. For the aminopeptidase activity,
on the other hand, exchange of Lys565 for an Ala or Met mostly affected the Michaelis
constant, with only marginal effects on the Vmax value, whereas exchange of Lys565 for an
Arg affected neither Vmax nor Km. Thus, for [K565A]LTA4H, Vmax and Km were 78 and 483%,
respectively, of the wild type enzyme and for [K565M]LTA4H the corresponding values were
58 and 645%, respectively. For [K565R]LTA4H, which retains the positive charge, values of
Vmax and Km remained at 108 and 82%, respectively (Table II). The effects on the Michaelis
constants were also reflected in the specificity constants of [K565A]- and [K565M]LTA4H,
which were reduced to 16 and 9%, respectively, but increased minimally in [K565R]LTA4H
to 132%. It should be noted that Lys565 is part of a salt bridge involving Glu533 and that
effects of mutations may, at least to some extent, be explained by a disturbance of these
charge interactions.
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Control mutants [R563K/K565R]- and [E560Q]LTA4H
As expected, the double cross-over mutant [R563K/K565R]LTA4H, yielded an enzyme
devoid of epoxide hydrolase activity, as seen in [R563K]LTA4H. However,
[R563K/K565R]LTA4H exhibited a reduced peptidase activity, exhibiting a Vmax and Km of
18 and 47%, respectively, of wild type enzyme. The specificity constant for the double cross-
over mutant attained 39% of that of wild type enzyme.
The control mutation close to Arg563 and Lys565 did not cause any major effects on
enzyme catalysis. For the epoxide hydrolase activity, [E560Q]LTA4H exhibited a Vmax and
Km of 61 and 60%, respectively, of wild type enzyme (Table II). As a consequence, the
specificity constant was not significantly changed. For the aminopeptidase activity,
[E560Q]LTA4H exhibited a Vmax and Km of 39 and 109%, respectively, of wild type enzyme
(Table II). The kcat/Km value for [E560Q]LTA4H was reduced to 36%.
Analysis of LTA4 binding strength by enzyme inhibition assays
Since all mutants in position 563 lacked epoxide hydrolase activity, inhibition assays against
the residual aminopeptidase activity were carried out to probe the effects of mutations on the
binding of LTA4. For [R563K]- and [R563M]LTA4H, the hydroxamic acid inhibitor
exhibited a Ki of 0.1 and 0.2 µM, as compared to 0.01 µM for wild type enzyme. In contrast,
no significant difference in Ki could be detected between [R563K]-, [R563M]- and wild type
LTA4H when employing LTA4 as an inhibitor of the aminopeptidase activity (Table III).
Thus, the Ki for LTA4 (0-10 µM) was determined to 7.5, 5.5 and 8 µM for [R563K]-,
[R563M]- and wild type LTA4H, respectively. These values are similar to previously reported
Km values for LTA4 (5-30 µM), suggesting that the substrate binds equally strong and in
similar conformations to both mutated and wildtype LTA4H.
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Crystal structure of [R563A]LTA4H
The overall structure of [R563A]LTA4H is very similar to that of wild type LTA4H (26),
with an overall r.m.s. difference of all Cα atoms of 0.39 Å, and for all protein atoms 0.7 Å
between the two structures. The structure of [E271Q]LTA4H (25) was used to calculate initial
electron density maps. In the initial Fobs-Fcalc electron density map, negative densities were
observed around the side-chain of residue 563, verifying the correct exchange of the original
Arg for an Ala. In the finally refined structure of [R563A]LTA4H, only the loop between
residues 175 and 185 differs from its conformation in the wild type enzyme, the reason for
which could be differences in crystal packing between the two different space groups. Both
wild type LTA4H and [R563A]LTA4H were co-crystallized with bestatin. Whereas in the
Fobs-Fcalc electron density map of wild type LTA4H bestatin was readily identified, no
features interpretable as this tight-binding inhibitor were found in the Fobs-Fcalc electron
density map of [R563A]LTA4H. The local environment of the region of mutation exhibits a
slight structural modification, in which the loss of the arginyl side-chain gives rise to an
increased, locally available, space which displaces the Nζ of Lys565 about 2.5 Å towards the
initial position of Arg563 (Fig. 2).
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Discussion
The biological effects of LTB4 are signaled via the BLT1 receptor present on the surface of
immuno-competent cells, e.g. peripheral leukocytes, including neutrophils and eosinophils, as
well as peritoneal macrophages (37,38). The chemical properties of LTB4 that are crucial for
agonistic action at the BLT1 receptor include a ∆6−cis-∆8-trans-∆10-trans double bond
geometry and an R-configuration of the hydroxyl group at C12, as determined by structure-
activity studies (21-23). Previous work on mutated enzymes and non-mammalian LTA4H
have indicated that a precise alignment of LTA4 in the active site is also very important for
catalysis and formation of the proper chemistry in the product LTB4 (18,39-41). This
conclusion is further corroborated by the crystal structure of LTA4H, which suggests that the
critical ∆6-cis double bond in LTB4 may be governed by the specific alignment of LTA4 along
an L-shaped, putative substrate-binding tunnel (26). Apparently, recognition of the substrate
LTA4 precedes these events and must be a key factor for successful catalysis.
In the X-ray crystal structure of LTA4H, two positively charged residues, Arg563 and
Lys565, are located in a wide portion of the putative substrate binding cavity, near its
entrance, and in the vicinity of the carboxyl group of bestatin. To explore the potential
involvement of Arg563 and Lys565 in the recognition, binding and turnover of substrates, we
carried out a combined mutational and structural analysis of these residues.
Arg563 is critical for turnover of LTA4
Mutations of Arg563 resulted in complete loss of catalytic function. Surprisingly, the
conservative replacement of Arg for Lys, which preserves the positive charge, also abolished
enzyme function, indicating that Arg563 plays a critical role in substrate turnover (Table II).
This conclusion is supported by the structure of [R563A]LTA4H which did not reveal any
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structural alterations that could explain the complete loss of catalytic activity (Fig. 2). In
addition, the mutant [R563K]LTA4H exhibited a significant residual aminopeptidase activity
indicating that no major structural changes had occurred at the active site.
Mutations of Arg563 do not compromise binding strength for LTA4
Since [R563K]LTA4H did not display any epoxide hydrolase activity, values of Km could not
be determined as a measure of LTA4 binding. Instead, we used an indirect approach, taking
advantage of the residual peptidase activity of [R563K]- and [R563M]LTA4H, to evaluate the
contribution of Arg563 to the binding strength between LTA4 and the protein. Thus, we
analyzed the effects of mutations on the inhibitory efficiency of LTA4 itself and a tight-
binding hydroxamic acid inhibitor that was designed as a mimic of LTA4 (42). The structure
of LTA4H complexed with this inhibitor has been determined (28). In the structure, the
inhibitor occupies almost entirely the proposed LTA4 binding site, and its carboxyl end makes
a direct salt bridge with Arg563. For [R563K]- and [R563M]LTA4H, the Ki values for LTA4
were 7.5 and 5.5 µM, respectively, as compared to 8 µM for the wild type enzyme, indicating
that exchange of Arg for Lys or Met did not affect substrate binding strength. This finding
may be interpreted in two ways, either Arg563 does not contribute that much to the overall
binding strength between LTA4 and the protein, and/or the carboxylate tail of LTA4 binds in
alternative conformations, with similar strength, in the mutated enzymes. The corresponding
Ki values for the hydroxamic acid were 0.1, 0.2 and 0.01 µM for [R563K]-, [R563M] and
wild type LTA4H, respectively. The reduced efficiency of the hydroxamic acid in
[R563K]LTA4H most probably reflects an increased space between the zinc and the positive
charge of Lys, which in turn may affect zinc-hydroxamate interactions. In addition, an Arg
side-chain provides two bonds for carboxylate interaction, which increases the precision of
binding, whereas a Lys only provides a single bond (43). Removing the charge by
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replacement of Arg for Met only caused a marginal additional reduction of inhibitor potency.
Taken together, these data demonstrate that the loss of epoxide hydrolase activity in
[R563K]LTA4H is not related to a decreased binding strength between substrate and protein
and further corroborate the notion that Arg563, in an indirect manner (see below), is required
for successful turnover of LTA4 into LTB4.
Arg563 is an atypical recognition site for the carboxyl group of LTA4
The original proposal that arginyl residues are common anion recognition sites in many, if not
all, enzymes utilizing anionic substrates is now a well-established fact in enzymology (44). It
holds true also for enzymes involved in fatty acid metabolism, e.g. cytochrome p450BM-3
(45) and central enzymes in the eicosanoid cascade such as COX-1 and COX-2 (46-50). The
usual role of the positively charged residue is to guide the incoming substrate and provide
firm anchoring to the protein. Thus, mutations of carboxylate recognition sites normally lead
to a reduced activity due to increased Km for the substrate. In this respect, LTA4H appears to
be an unusual example of an enzyme that is critically dependent on the Arg residue for
enzyme function, rather than achieving sufficient substrate binding strength.
Arg563 ensures precision in LTA4 alignment at the active site
It has been known since long that esterified LTA4 is not a substrate for LTA4H (51), which
has sometimes been interpreted as the result of steric hindrance at the active site. This strong
specificity for the free acid of LTA4 has also been taken as evidence for a catalytic
mechanism involving a substrate-catalyzed, carboxylic acid-induced, activation of the epoxide
moiety as an initial step in the enzymatic reaction (29). From the present data, however, it
seems clear that the absolute requirement of a free C1 carboxylate is best explained by the
need of a precise positioning of the substrate, via Arg563, to guarantee optimal alignment
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necessary for catalysis (Fig. 3A). The neighboring residue, Lys565, which is also located such
that it could well be involved in carboxylate recognition, seems to have little, if any, role in
the binding and turnover of LTA4 (Table II). Thus, the epoxide hydrolase activities of
[K565R]-, [K565A]- and [K565M]LTA4H were not significantly decreased.
The C-terminal anchoring of peptide substrates appear to involve both Arg563 and Lys565
Mutations of Arg563 reduced the aminopeptidase activity of LTA4H albeit to a variable
degree. The activity of [R563A]LTA4H against Ala-p-NA was barely detectable, whereas
[R563K]- and [R563M]LTA4H were catalytically less compromised with a significant
residual activity of 6.4 and 20%, respectively, as compared to the wild type enzyme (Table
II). These effects suggest that Arg563 is a carboxylate binding site also for peptide substrates
(Fig. 3B). However, unlike the anchoring of LTA4 in the epoxide hydrolase reaction, Arg563
seems to be assisted by Lys565 in the recognition of peptides. Thus, when compared to wild
type enzyme, the turnover of Ala-p-NA by [K565A]-, [K565M]- and [K565R]LTA4H was
only slightly affected whereas a distinct increase in the Michaelis constant was observed for
[K565A]- and [K565M]LTA4H, i.e. the two mutants lacking positive charge (Table II).
The natural peptidase substrate(s) of LTA4H is currently not known but the enzyme can
hydrolyze a variety of compounds containing a peptide or amide bond. One of the best and
most frequently used substrates is Ala-p-NA, which is used as a model substrate for the
aminopeptidase reaction. Although molecular docking experiments indicate that Ala-p-NA
aligns in the catalytic pocket with the nitro-group in a similar orientation to that of the
carboxylate of tripeptide substrates (25), these two functional groups are not equivalent. The
carboxylate has a negative net charge, whereas the nitro-group, which exhibits a delocalized
charge, has a net charge of zero. Therefore, Ala-p-NA is not a perfect aminopeptidase
substrate, which limits our possibilities to predict the behavior of natural peptide substrate(s).
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However, crystallographic data offer further support for our conclusions. In the crystal
structure of wild type LTA4H, the tight-binding, competitive, inhibitor bestatin was included.
Bestatin is also a dipeptide mimetic and in the structure, the carboxylate moiety of the
molecule appears to interact with Arg563 and Lys565 via an intervening water molecule,
suggesting that the C-terminus of a dipeptide substrate may bind in a similar manner (26).
Also, in the crystal structure of LTA4H complexed with captopril, a direct interaction was
seen between this inhibitor and Arg563 (28). Bestatin was included in the crystallization of
[R563A]LTA4H, but in the 3D-structure the compound could not be identified at the active
site, suggesting absence or low occupancy of the inhibitor, possibly due to loss of binding to
Arg563. Taken together, the mutagenetic and crystallographic data indicate that Arg563 is
responsible for the basal binding of the C-terminus of a peptide substrate required for an
alignment compatible with catalysis. Lys565, on the other hand, seems to contribute
significant binding strength and thus cooperates with Arg563 to achieve optimal conditions
for efficient substrate turnover (Fig. 3B).
The carboxylate recognition site, Arg563/Lys565, is a unique feature of LTA4H
LTA4H belongs to the M1 family of zinc metallopeptidases, which is a subfamily of the
broader MA clan of “thermolysin-like” metalloproteases (52). A common structural feature in
this large group of enzymes is the zinc binding motif HEXXH-(X)18 -E (53) and in LTA4H,
the catalytic zinc is coordinated by His295, His299 and Glu318 (54). More specifically, the
M1 family includes enzymes such as aminopeptidase A (EC 3.4.11.7, APA), aminopeptidase
B (EC 3.4.11.6, APB) and aminopeptidase N (EC 3.4.11.2, APN), all of which share a
conserved GXMEN motif. This signature harbors a Glu residue acting as an N-terminal
recognition site for peptide substrates. In LTA4H, the corresponding Glu271 is also critical
for the epoxide hydrolase reaction, presumably in the early activation and opening of the
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epoxide moiety (25). In the tertiary structure, most catalytic residues of LTA4H, e.g. Glu271,
Glu296, Asp375 and Tyr383 are located within the central catalytic domain. This domain is
structurally very similar to thermolysin and is possibly a common structural feature for the
entire MA clan of metalloproteases, which include the M1 metallopeptidases (55). However,
unlike the other catalytic residues, Arg563 and Lys565 are located within the C-terminal
domain, which seems to be the segment of the protein that is most typical for LTA4H (26).
Considering the role of Glu271 as an anchor for the α-amino group of a peptide substrate
(25), the Arg/Lys site may limit the length of the substrate and thus becomes an important
determinant for the tripeptide specificity of LTA4H. Concerning the evolutionary
preservation of Arg563 and Lys565, these residues are highly conserved among LTA4H
homologues across a wide range of species, but show less conservation between other
members of the M1 family (Fig. 4). However, only one of the non-mammalian homologues,
viz. S. cerevisiae LTA4H, has been shown to possess an epoxide hydrolase activity (41) and
the enzyme from C. elegans has even been reported to lack this activity (56). In other
mammalian M1 metallopeptidases such as APA and APN, this site is not conserved since the
domain in which these residues are found is replaced by another domain sequence.
Among other M1 metallopeptidases, APB seems to exhibit the highest degree of
identity with LTA4H over the C-terminal domain and also carries a Lys/Lys site, suggesting
that APB may well be a tripeptidase. This hypothesis agrees well with earlier work with
human and rat APB, showing that di- and tripeptides are good substrates whereas
oligopeptides are not hydrolyzed (57). In any event, our data suggest that evolution has
developed Arg563 and Lys565 to carry out a fundamental function in carboxylate recognition
as well as catalytic turnover of both lipid and peptide substrates, in the common active center
of LTA4H.
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Acknowledgements.
We thank personnel at beamline I711 of MAX-Lab, Lund for help during data collection and
Eva Ohlson for technical assistance. The work was funded by the Swedish Medical Research
Council (O3X-10350), The European Union (QLG1-CT-2001-01521), The Swedish
Foundation for Strategic Research (fellowships to P.C.R. & M.A.), The Wallström
Foundation, Konung Gustav V:s 80-Årsfond, the Swedish Natural Sciences Research Council
(M.T.), and AFA Health Insurances.
Coordinates
Coordinates of [R563A]LTA4H have been deposited in the Protein Data Bank, accession
code 1SQM.
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Figure legends
FIG. 1. Stereo view of LTA4H, spatial relationships between residues and the substrate
LTA4 at the catalytic zinc site
In this stereo image, wild type LTA4H is depicted in a ball- and stick model. LTA4 was
modeled into the putative hydrophobic, L-shaped, catalytic pocket. The carboxyl group of
LTA4 faces the side-chains of Arg563 and Lys565. The catalytic residues Glu271 (25) and
Asp375 (20) are shown, and the catalytic zinc is depicted as a silver sphere. The figure was
generated with Swiss-PdbViewer (58) and POV-Ray (http://www.povray.org).
FIG. 2. Stereo view of the loop 562 - 566 of [R563A]LTA4H in Fobs-Fcalc electron density
For the calculation of Fcalc and αcalc, the atoms of loop 562-566 were excluded from the
calculations. The density was contoured at 3σ level. The picture was made using Molscript
(59), Glr (L. Esser & J. Deisenhofer, personal communications) and POV-Ray
(http://www.povray.org).
FIG. 3. Models for the catalytic mechanisms in LTA4H
(A) In the epoxide hydrolase reaction a water molecule is polarized by Glu271 and the
catalytic zinc (25), to promote an acid-induced opening of the epoxide. This reaction yields a
carbocation intermediate and finally a nucleophilic attack, guided by Asp375, occurs at C12
(20). In this reaction Arg563 serves in carboxylate recognition and substrate alignment.
Dotted lines indicate interactions between important groups. (B) In the aminopeptidase
reaction, Glu271 is involved in N-terminal recognition (25), Glu296 and the catalytic zinc act
as base catalyst and Tyr383 functions as proton donor (60,61). Here, Arg563 and Lys565
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serve together in carboxylate recognition. Dotted lines indicate interactions between
important groups. See text for further details.
FIG. 4. Sequence alignment of members of the M1 family of the MA clan of
metallopeptidases
The figure shows residues subjected to mutational analysis, as well as the three zinc ligands in
members of the M1 family of metallopeptidases. Note the strict conservation of the zinc
ligands (His295, His299 and Glu318), as opposed to the carboxylate recognition site. The
sequence alignment was created using CLUSTALW at Biology WorkBench 3.2
(http://workbench.sdsc.edu).
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TABLE I. Data collection and refinement statistics.
Data collection statistics
Diffraction limit (Å) 2.3
Wavelength (Å) 0.968
Completeness (%) 99.7 (99.7)
Mean I/σ (I) 5.3 (3)
Multiplicity of observation 4.7 (4.7)
Rmergea (%) 9.2 (23.1)
Refinement statistics
R-factorb (%) 18. 8
Rfreec (%) 23.9
R.m.s.d. in bond distance (Å) 0.0079
R.m.s.d. in bond angle (°) 1.44
a Rmerge = ΣhΣi|Ii (h) - I(h)|/ ΣhΣiIi (h), where Ii (h) is the ith measurement of reflection h and
I(h) is the weighted mean of all measurements of h.
b R-factor = Σh|Fobs (h) – Fcalc (h)|/ ΣhFobs (h), where
Fobs and Fcalc are the observed and calculated factor amplitudes, respectively.
c Rfree is the R-factor calculated for the test set of reflections (3.3 %) which are
omitted during the refinement process.
Data for the highest resolution shell (2.3–2.42 Å) are shown within parantheses.
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TABLE II. Kinetic parameters for the aminopeptidase and epoxide hydrolase activities
Mutated recombinant enzymes were expressed in E. coli, purified by affinity chromatography
and assayed for the aminopeptidase and epoxide hydrolase activity. Apparent kinetic
constants are expressed in % of those of wild type enzyme. Each data point was calculated as
mean, ± s.e., triplicate determinations. n.d. = not detectable.
Epoxide hydrolase activity
(% of WT) Aminopeptidase activity
(% of WT)
Mutant Vmax Km kcat/Km Vmax Km kcat/Km
Wild type 100 ± 7 100 ± 25 100 100 ± 2 100 ± 5 100
E560Q 61 ± 3 60 ± 12 100 39 ± 4 109 ± 10 36
R563K n.d. n.d. 6.4 ± 0.9 58 ± 15 11
R563A n.d. n.d. 0.3 ± 0.01 6.2 ± 1.6 5
R563M n.d. n.d. 20 ± 2 179 ± 37 11
K565R 95 ± 12 104 ± 33 92 108 ± 17 82 ± 21 132
K565A 31 ± 4 14 ± 16 218 78 ± 12 483 ± 110 16
K565M 32 ± 2 33 ± 12 95 58 ± 12 645 ± 173 9
R563K/K565R n.d. n.d. 18 ± 3 47 ± 14 39
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TABLE III. Inhibition assays
Ki (µM)
Inhibitor WT R563K R563M
Hydroxamic acid 0.01 0.1 0.2
LTA4 8 7.5 5.5
Values for inhibition constants (Ki) are in µM and were determined by Dixon plots. The
hydroxamic acid-like inhibitor was used in concentrations between 0.01 and 0.3 µM. LTA4
was used in concentrations between 0 and 10 µM.
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and Jesper Z. HaeggströmPeter C. Rudberg, Fredrik Tholander, Martina Andberg, Marjolein M.G.M. Thunnissen
for the epoxide hydrolase and aminopeptidase substratesLeukotriene A4 hydrolase: Identification of a common carboxylate recognition site
published online April 12, 2004J. Biol. Chem.
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