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LEUKOTRIENE A4 HYDROLASE/AMINOPEPTIDASE,
The gatekeeper of chemotactic leukotriene B4 biosynthesis
Jesper Z. Haeggström
Department of Medical Biochemistry and Biophysics, Division of Chemistry 2,
Karolinska Institutet, S-171 77 Stockholm, SWEDEN.
Correspondence:
Jesper Z. Haeggström
Department of Medical Biochemistry and Biophysics, Division of Chemistry 2,
Karolinska Institutet, S-171 77 Stockholm, Sweden
Telephone: +46-8-52487612
Fax: +46-8-736 0439
E-mail: [email protected]
JBC Papers in Press. Published on August 31, 2004 as Manuscript R400027200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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The leukotrienes (LTs) are a family of lipid mediators that play important roles in a
variety of allergic and inflammatory reactions (1, 2). These molecules are formed by
leukocytes and are divided into two classes, the spasmogenic cysteinyl-leukotrienes and
LTB4, which is a classical chemoattractant that triggers adherence and aggregation of
leukocytes to the endothelium at nM concentrations. Recent data also indicate that LTB4 is a
chemoattractant for T-cells, creating a functional link between early innate and late adaptive
immune responses to inflammation (3-5). In addition, LTB4 participates in the host-defense
against infections (6) and is a key mediator of PAF-induced lethal shock (7). Because of these
powerful biological effects, LTB4 is regarded as an important chemical mediator in a variety
of acute and chronic inflammatory diseases, e.g., nephritis, arthritis, dermatitis, and chronic
obstructive pulmonary disease (8). Moreover, only recently, several lines of pharmacological,
morphological, biochemical, and genetic evidence have been gathered implicating LTs, in
particular LTB4, as a mediator of vascular inflammation and arteriosclerosis (9). This article
gives an overview of the biochemical, structural and catalytic properties of LTA4 hydrolase
(LTA4H), which catalyzes the final and committed step in LTB4 biosynthesis.
LTA4 hydrolase is a key enzyme in the 5-lipoxygenase pathway.
In cellular biosynthesis of LTs, 5-lipoxygenase (5-LO), assisted by 5-LO activating protein
(FLAP), converts arachidonic acid into the unstable epoxide LTA4, which in turn may be
enzymatically conjugated with GSH to form LTC4, the parent compound of the cysteinyl-
leukotrienes, or hydrolyzed into LTB4 by LTA4H (Fig. 1). Leukotrienes can also be formed
via transcellular routes, where LTA4 is donated from an activated leukocyte to a recipient cell
for further metabolism by downstream enzymes, a process that was recently shown to occur in
vivo (10). LTB4 signals via a specific, high-affinity, G-protein coupled receptor (BLT1) (11).
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In addition, a second receptor for LTB4 (BLT2) has been discovered, the functional role of
which is presently not known (12). Interestingly, LTB4 is also a natural ligand of the PPARα
class of nuclear receptors and has been suggested to play a role in lipid homeostasis (13).
Leukotriene A4 hydrolase is a zinc dependent epoxide hydrolase and aminopeptidase.
LTA4H is a monomeric soluble protein that is widely distributed and has been purified from
several mammalian sources (14). It resides in the cytosol, although nuclear localization has
also been recently reported (15). The cDNAs encoding the human, mouse, rat, and guinea-pig
enzymes have been cloned and sequenced. The proteins contain 610 amino acids, excluding
the first Met, and the calculated molecular mass of the human enzyme is 69,153 Da. The
primary, secondary and tertiary structures of LTA4H bear no resemblance to soluble
xenobiotic epoxide hydrolase, although this enzyme also accepts LTA4 as substrate (16).
Human LTA4H exists as a single copy gene with a size of > 35 kbp on chromosome 12q22
(17). The coding sequence is divided into 19 exons ranging in size from 24 to 312 bp and the
5’ upstream region (approx. 4 kbp) contains a phorbol-ester response element (AP-2) and two
xenobiotic-response elements (XRE), the functional significance of which are presently
unknown.
LTA4H is highly substrate specific. Besides LTA4, only the double bond isomers LTA5
and LTA3 are turned over by LTA4H, albeit at a low rate (18, 19). Typically, LTA4H is
inactivated and covalently modified by its substrate LTA4 during catalysis (18, 19). Using
differential peptide mapping and site directed mutagenesis, Tyr-378 has been identified as the
site of attachment between lipid and protein and the catalytic properties of the mutated protein
[Y378F]LTA4H suggest that this residue may assist in the proper alignment of LTA4 in the
substrate-binding pocket (20, 21). Although it has been reported that LTA4H may be
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phosphorylated at Ser-415 under certain conditions (22), no other specific post-translational
modifications seem to occur.
Sequence comparisons between LTA4H and several zinc hydrolases, e.g., aminopeptidase
M and thermolysin, led to the discovery of a catalytic zinc site with the signature HEXXH-
(X)18-E and subsequent analysis by atomic absorption spectrometry revealed the presence of
one mol of zinc/mol of protein (Fig. 2). As suspected from the homology to zinc proteases,
LTA4H was found to possess a peptide cleaving activity. Unlike the epoxide hydrolase
activity, i.e., the transformation of LTA4 into LTB4, the aminopeptidase activity is activated
by monovalent anions and albumin. It accepts a variety of substrates and certain arginyl di-
and tripeptides as well as p-nitroanilide derivatives of Ala and Arg are hydrolyzed with high
efficiencies (23). Although it has never been experimentally verified, it is generally assumed
that the aminopeptidase activity is involved in the processing of peptides related to
inflammation and host-defense.
The zinc site and catalytic residues
The three zinc binding ligands of the signature HEXXH-(X)18-E corresponds to His-295, His-
299, and Glu-318 in LTA4H and mutation of any of these residues leads to loss of the metal
and both catalytic activities (24). The conserved Glu-296 in the motif HEXXH was identified
as the critical general base of the peptidase reaction without any apparent function in the
epoxide hydrolase reaction (25, 26). Furthermore, sequence comparisons with aminopeptidase
N, suggested that Tyr-383 might act as a proton donor in peptidolysis. Indeed, mutation of
this residue resulted in selective abrogation of the aminopeptidase activity, thus supporting a
catalytic role for Tyr-383 (27).
Several candidate catalytic residues were identified from the crystal structure of LTA4H
(see below). Glu-271 is a component of a GXMEN motif, which is conserved among
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members of the M1 family of metallopeptidases and proposed to play a role in peptide
substrate binding (28). We used mutagenesis and crystallography to detail the role of
individual residues within the GXMEN motif and found that Glu-271 is indeed required for
the peptidase activity and, unexpectedly, also for the epoxide hydrolase activity (29). With the
same technique, it was demonstrated that Asp-375, located in a putative LTA4 binding pocket,
is required for hydrolysis of LTA4 into LTB4 but not for the aminopeptidase activity (30). In
addition, analysis of an Arg/Lys couple located at the entrance of the catalytic zinc site
demonstrated that Arg-563 is a carboxylate recognition site that plays a key role in the
epoxide hydrolase reaction and binds the C- terminus of peptide substrates, assisted by Lys-
565 (31).
Crystal structure of LTA4 hydrolase.
The structure of LTA4H in complex with the competitive inhibitor bestatin has been
determined at 1.95 Å resolution (32). The protein molecule is folded into three domains: N-
terminal, catalytic, and C-terminal, that are packed in a flat triangular arrangement with
approximate dimensions 85 x 65 x 50 Å3 creating a deep cleft in between (Fig. 3). The N-
terminal domain has a large concave and hydrophobic surface area and is structurally similar
to bacteriochlorophyll a (33). The fold of the catalytic domain is very similar to that of
thermolysin although the sequence identity is only about 7% over the corresponding
polypeptide chains. The C-terminal domain has structural features resembling a so called
armadillo repeat or HEAT region, which in turn suggests that it may take part in protein-
protein interactions (34).
The zinc site is located at the bottom of the interdomain cleft. As predicted, the metal is
bound to His-295, His-299, and Glu-318, with bestatin as the fourth ligand in a pentavalent
coordination. In the vicinity of the prosthetic zinc, the catalytic residues Glu-271, Glu-296
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and Tyr-383 are located. Behind the pocket occupied by the phenyl ring of bestatin there is an
L-shaped hydrophobic cavity approximately 6-7 Å wide, which stretches 15 Å deeper into the
protein (Fig 3). One patch of the cavity is hydrophilic, with Gln-134, Asp-375 and the
hydroxyl of Tyr-267 clustering together. This cavity was probed by structural determination
of complexes between LTA4H and specific, active site-directed, inhibitors, some of which
have been designed as LTA4 mimics (35). Indeed, the hydrophobic tail of the inhibitors,
corresponding to the fatty acid backbone of LTA4, is buried into the narrow hydrophobic
pocket, strongly indicating that it functions as a substrate-binding cavity (Fig. 4).
Proposed mechanism of the epoxide hydrolase reaction.
The stereochemistry at C12 and double bond geometry are key structural determinants for the
biological activity of LTB4. Consequently, the role of LTA4H during enzymatic hydrolysis of
LTA4 into LTB4 is to generate the 12R epimer of the hydroxyl group and to form the ∆6-cis-
∆8-trans-∆10-trans configuration of the conjugated triene. This reaction is unique and requires
control of the stereospecific introduction of a hydroxyl group at a site (C12) distant from the
reactive epoxide moiety (C5-C6). The crystal structure gives several important clues as to
how LTA4H can execute its sophisticated chemistry.
If LTA4 is modeled into the hydrophobic pocket such that the 5,6-epoxide moiety is bound
to Zn2+, then C-7 to C-20 of the fatty acid backbone of LTA4 fits snugly into the deeper
cavity, adopting a bent conformation (Fig. 4). Furthermore, the C-1 carboxylate can make
direct electrostatic interactions with the positive charge of Arg-563. This crucial interaction
will also assure perfect substrate alignment required for catalysis (31), in agreement with the
fact that the free carboxylic acid of LTA4 is required for catalysis (19, 36). Furthermore, the
catalytic zinc as well as Glu-271 will be proximal to the labile allylic epoxide, suggesting that
they will polarize a water molecule and promote an acid induced activation and opening of the
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epoxide ring (Fig. 4). A carbocation will be generated, according to an SN1 reaction, whose
charge is delocalized over the conjugated triene system (C-6 to C-12), leaving the planar sp2
hybridized C-12 open for nucleophilic attack from either side of the molecule. In this model,
Asp-375 would direct a water molecule for attack at C-12 and thus control the positional and
stereospecific insertion of the 12R-hydroxyl group in LTB4, in agreement with mutational
data (30).
Moreover, the shape and curvature of the LTA4 binding cavity also suggest the chemical
strategy for creation of the 6-cis double bond in LTB4. Since there is free rotation between C-
6 and C-7 of LTA4, the enzyme may keep this bond in a “pro-cis” configuration in the
transition state, which would promote the formation of a cis double bond from the carbocation
intermediate (Fig. 4). The entire modeled LTA4 molecule would then adopt a bent shape that
fits very well with the architecture of the binding pocket. Hence, the critical double bond
geometry at ∆6 in LTB4 seems to be controlled by the exact binding conformation of LTA4 at
the active site.
Proposed mechanism for the aminopeptidase activity.
In agreement with what has been discussed for thermolysin, the peptide cleaving activity of
LTA4H most likely proceeds according to a general base mechanism (37). Thus, the catalytic
zinc is complexed to its three amino acid ligands and an activated water molecule. The water
is displaced from the zinc atom by the carbonyl oxygen of the substrate, which in turn gets
anchored to the active site via its N-terminal α-amino group binding to Glu-271. In this role,
Glu-271 will stabilize the transition state and also contribute to the enzyme’s exopeptidase
specificity (Fig. 4). The water molecule is simultaneously polarized by the carboxylate of
Glu-296 to promote an attack on the carbonyl carbon of the scissile peptide bond. At the same
time, a proton is transferred to the nitrogen of the peptide bond by Tyr-383.
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Two catalytic activities exerted via specific but overlapping active sites.
Compilation of information from biochemical studies, mutational analysis and X-ray
crystallography leads to the conclusion that the two enzyme activities of LTA4H are exerted
via distinct and yet overlapping active sites (Figs. 1 and 4). Thus, certain residues are
specifically required for the aminopeptidase reaction, i.e., Glu-296 and Tyr-383, whereas
Asp-375 is critical only for the epoxide hydrolase reaction. On the other hand, Glu-271, Arg-
563, and the zinc atom are necessary for both catalyses. In fact, Glu-271 is a unique example
of a residue that is shared between two catalytic machineries, yet carrying out a separate
chemistry in each of the two enzyme reactions.
Molecular evolution of LTA4 hydrolase.
Based on its zinc signature and aminopeptidase activity, LTA4H is classified as a member of
the M1 family of zinc metallopeptidases, which includes enzymes such as aminopeptidase A,
aminopeptidase B, and aminopeptidase N (28). However, the epoxide hydrolase activity
appears to be unique for LTA4H and has not been detected with certainty in any human
homologue, although conflicting data have been reported for aminopeptidase B (38, 39).
LTA4H is present in several lower vertebrates including fish and frogs but not in lower
species (40, 41). For instance, aminopeptidase 1 from Caenorhabditis elegans, which is 45%
identical (63% similar) at the amino acid level to mammalian LTA4H, fails to hydrolyze
LTA4 into LTB4 (42). On the other hand, an LTA4H that is 39% identical (53% similar) to
the human enzyme, has been cloned and characterized from yeast, Saccharomyces cerevisiae
(43). The S. cerevisiae LTA4H is a zinc leucyl aminopeptidase with a primitive epoxide
hydrolase activity against LTA4. Furthermore, binding of LTA4 to the active site leads to
inactivation of the epoxide hydrolase activity and strong activation of the peptidase activity.
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Together, these data suggest that LTA4H has developed from an ancestral aminopeptidase,
which initially possessed an allosteric lipid-binding site. During evolution, the architecture
was remodeled into an active site accommodating LTA4. Subsequent structural optimizations
further improved substrate alignment, and finally allowed efficient catalysis and formation of
LTB4.
LTA4 hydrolase, an attractive target for structure-based drug design.
The specific roles of LTB4 in acute and chronic inflammation have been mapped and
corroborated by several animal models of perturbed biosynthesis or signaling, targeting all
components of the metabolom (7, 44-48). Together, this wealth of in vivo data points to
LTA4H as a potential target for development of anti-inflammatory drugs. Moreover, the
pharmacological interest has been increased even further by the recent observations of
increased protein expression in esophageal cancer and graft-versus-host disease following
hematopoietic stem cell transplantation (49, 50).
Bestatin and captopril, inhibitors of aminopeptidases and angiotensin converting enzyme,
respectively, also inhibit LTA4H (51). In addition, ω-[(ω-arylalkyl)aryl]alkanoic acids and
kelatorphan, a known inhibitor of enkephalin degrading enzymes, are potent inhibitors (52,
53).
Several academic and industrial laboratories have developed more powerful and selective
compounds. An α-keto-β-amino ester, a thioamine, and a hydroxamic acid were synthesized
and found to be effective, tight-binding inhibitors with IC50 values in the low µM to nM
range (54, 55). Other series of potent inhibitors have also been developed by Searle, in
particular SC-57461A, 3-[methyl[3-[4-(phenylmethyl)-phenoxy]propyl]-amino propanoic
acid which blocks ionophore-induced LTB4 production in human whole blood with an IC50
of 49 nM, is orally active, and blocks arachidonic acid induced ear edema in the mouse (56,
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57). With the 3D-structure of LTA4H at hand, it will now be possible to conduct rational
structure-based drug design to tailor potent and selective inhibitors with desired
pharmacogical properties.
Acknowledgments
This work was financially supported by the Swedish Medical Research Council (O3X-10350),
the European Union, AFA Health Foundation, and Konung Gustav V:s 80-Årsfond.
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Figure legends
Figure 1. Major cellular constituents of the LTB4 metabolom. Calcium induces translocation
of phospholipase A2 (cPLA2) from cytosol to the nuclear envelope, which is followed by
liberation of arachidonic acid (AA) from membrane phospholipids. 5-LO, present in cytosol
or nucleus, also translocates to the same compartment, allowing transformation of AA into
LTA4 in a reaction assisted by FLAP. LTA4 may in turn be hydrolyzed into LTB4, by
LTA4H, or converted into LTC4 by a specific synthase. Further details, see (58).
Figure 2. Model of structural and functional properties of LTA4H.
Figure 3. X-ray crystal structure of LTA4H.
Overall structure of LTA4H depicted as a ribbon diagram. The N-terminal domain is shown in
blue, the catalytic domain in green and the α-helical C-terminal domain in red. The deep
cavity formed between the domains is depicted as a mesh in magenta and is composed of a
wide central section connected to a narrow and deeper L-shaped cavity.
Figure 4. Proposed catalytic mechanisms of the bifunctional LTA4H. The yellow areas
indicate the active site with its wide open section and narrow, L-shaped, hydrophobic pocket.
(A) Model of the epoxide hydrolase activity. Assisted by the carboxylate of Glu-271, the
catalytic zinc polarizes a water molecule to promote an acid induced activation of the epoxide
to form a carbocation intermediate. Water is added at C12 in a stereospecific manner, directed
by Asp-375. The double bond geometry is controlled by the binding conformation of LTA4.
Arg-563 acts as a critical carboxylate recognition site. For further details, see text. (B) Model
of the aminopeptidase activity. The fourth ligand of the catalytic zinc is an activated water
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17
molecule that is displaced by a carbonyl group of the incoming tripeptide substrate. The α-
aminogroup of the substrate gets attached to Glu-271, acting as an N-terminal recognition site.
The water is polarized by the base, Glu-296, and attacks the peptide bond. Simultaneously, a
proton is donated from Tyr-383.
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cPLA2
FLAP
LTA4
LTC4LTB4
AA
2+Ca
NucleusAA
5-LO
LTC 4synthase
LTA4hydrolase
Cell membrane
Haeggström Figure 1
cPLA2
5-LO
5-LO
2+Ca2+Ca
2+Ca
2+Ca
2+Ca
2+Ca
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Ala-4-NA LTA4
LTB4Ala + 4-NA
Glu-296 Tyr-383 Tyr-378Asp-375
Arg-563Arg-563
Lys-565Glu-271
H O2
...
..
...
..
...
..
His-295
His-299
Glu-318
+-Cl
2+Zn
Haeggström Figure 2
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Haeggström Figure 3
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O O
O+
HO
H
2+
Zn
O
O-
D375
HO
H
2+
Zn
R1
O
O
O
NN
R2
R3
O
-
Y383
HO
H
A
B
E271
OO-
Arg563
NH
NH2
NH2+
Lys565
NH+3
Lys565
NH+3
E271
OO-
-
NH3+
E296
O
O
-H H
O
HArg563
NH
NH2
NH2+
Haeggström Figure 4
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Jesper Z. Haeggströmleukotriene B4 biosynthesis
Leukotriene A4 hydrolase/aminopeptidase, the gatekeeper of chemotactic
published online August 31, 2004J. Biol. Chem.
10.1074/jbc.R400027200Access the most updated version of this article at doi:
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