leukotriene a4 hydrolase/aminopeptidase, the

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



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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+



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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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When a correction for this article is posted•

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