Leukotriene A4 Hydrolase/Aminopeptidase, the Gatekeeper of Chemotactic Leukotriene B4 Biosynthesis

The leukotrienes (LTs) 1The abbreviations used are: LT, leukotriene; LTA4H, LTA4 hydrolase. are a family of lipid mediators that play important roles in a variety of allergic and inflammatory reactions (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2322) Google Scholar, 2Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3050) Google Scholar). 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 nanomolar 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 (3Goodarzi K. Goodarzi M. Tager A.M. Luster A.D. von Andrian U.H. Nat. Immunol. 2003; 4: 965-973Crossref PubMed Scopus (294) Google Scholar, 4Ott V.L. Cambier J.C. Kappler J. Marrack P. Swanson B.J. Nat. Immunol. 2003; 4: 974-981Crossref PubMed Scopus (240) Google Scholar, 5Tager A.M. Bromley S.K. Medoff B.D. Islam S.A. Bercury S.D. Friedrich E.B. Carafone A.D. Gerszten R.E. Luster A.D. Nat. Immunol. 2003; 4: 982-990Crossref PubMed Scopus (346) Google Scholar). In addition, LTB4 participates in the host defense against infections (6Bailie M.B. Standiford T.J. Laichalk L.L. Coffey M.J. Strieter R. Peters-Golden M. J. Immunol. 1996; 157: 5221-5224PubMed Google Scholar) and is a key mediator of platelet-activating factor-induced lethal shock (7Byrum R.S. Goulet J.L. Snouwaert J.N. Griffiths R.J. Koller B.H. J. Immunol. 1999; 163: 6810-6819PubMed Google Scholar). 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 (8Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1179) Google Scholar). 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 (9Jala V.R. Haribabu B. Trends Immunol. 2004; 25: 315-322Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). 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. In cellular biosynthesis of LTs, 5-lipoxygenase, assisted by 5-lipoxygenase-activating protein, 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 (10Fabre J.E. Goulet J.L. Riche E. Nguyen M. Coggins K. Offenbacher S. Koller B.H. J. Clin. Invest. 2002; 109: 1373-1380Crossref PubMed Scopus (80) Google Scholar). LTB4 signals via a specific, high affinity, G-protein-coupled receptor (BLT1) (11Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (853) Google Scholar). In addition, a second receptor for LTB4 (BLT2) has been discovered, the functional role of which is presently not known (12Yokomizo T. Kato K. Terawaki K. Izumi T. Shimizu T. J. Exp. Med. 2000; 192: 421-432Crossref PubMed Scopus (470) Google Scholar). Interestingly, LTB4 is also a natural ligand of the peroxisome proliferator-activated receptor α class of nuclear receptors and has been suggested to play a role in lipid homeostasis (13Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1210) Google Scholar). LTA4H is a monomeric soluble protein that is widely distributed and has been purified from several mammalian sources (14Haeggström J.Z. Am. J. Respir. Crit. Care Med. 2000; 161: 25-31Crossref Scopus (67) Google Scholar). It resides in the cytosol, although nuclear localization has also been reported recently (15Brock T.G. Maydanski E. McNish R.W. Peters-Golden M. J. Biol. Chem. 2001; 276: 35071-35077Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). 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 (16Argiriadi M.A. Morisseau C. Hammock B.D. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10637-10642Crossref PubMed Scopus (208) Google Scholar). Human LTA4H exists as a single copy gene with a size of >35 kbp on chromosome 12q22 (17Mancini J.A. Evans J.F. Eur. J. Biochem. 1995; 231: 65-71Crossref PubMed Scopus (51) Google Scholar). The coding sequence is divided into 19 exons ranging in size from 24 to 312 bp, and the 5′ upstream region (approximately 4 kbp) contains a phorbol ester response element (AP-2) and two xenobiotic response elements, the functional significance of which is 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 (18Evans J.F. Nathaniel D.J. Zamboni R.J. Ford-Hutchinson A.W. J. Biol. Chem. 1985; 260: 10966-10970Abstract Full Text PDF PubMed Google Scholar, 19Ohishi N. Izumi T. Minami M. Kitamura S. Seyama Y. Ohkawa S. Terao S. Yotsumoto H. Takaku F. Shimizu T. J. Biol. Chem. 1987; 262: 10200-10205Abstract Full Text PDF PubMed Google Scholar). Typically, LTA4H is inactivated and covalently modified by its substrate LTA4 during catalysis (18Evans J.F. Nathaniel D.J. Zamboni R.J. Ford-Hutchinson A.W. J. Biol. Chem. 1985; 260: 10966-10970Abstract Full Text PDF PubMed Google Scholar, 19Ohishi N. Izumi T. Minami M. Kitamura S. Seyama Y. Ohkawa S. Terao S. Yotsumoto H. Takaku F. Shimizu T. J. Biol. Chem. 1987; 262: 10200-10205Abstract Full Text PDF PubMed Google Scholar). 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 (20Mueller M.J. Wetterholm A. Blomster M. Jörnvall H. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8383-8387Crossref PubMed Scopus (49) Google Scholar, 21Mueller M.J. Blomster M. Opperman U.C.T. Jörnvall H. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5931-5935Crossref PubMed Scopus (48) Google Scholar). Although it has been reported that LTA4H may be phosphorylated at Ser-415 under certain conditions (22Rybina I.V. Liu H. Gor Y. Feinmark S.J. J. Biol. Chem. 1997; 272: 31865-31871Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), 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 HEXXHX18E, and subsequent analysis by atomic absorption spectrometry revealed the presence of 1 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 (23Orning L. Gierse J.K. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 11269-11273Abstract Full Text PDF PubMed Google Scholar). 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 three zinc binding ligands of the signature HEXXHX18E 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 (24Medina J.F. Wetterholm A. Rådmark O. Shapiro R. Haeggström J.Z. Vallee B.L. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7620-7624Crossref PubMed Scopus (92) Google Scholar). 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 (25Wetterholm A. Medina J.F. Rådmark O. Shapiro R. Haeggström J.Z. Vallee B.L. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9141-9145Crossref PubMed Scopus (75) Google Scholar, 26Minami M. Bito H. Ohishi N. Tsuge H. Miyano M. Mori M. Wada H. Mutoh H. Shimada S. Izumi T. Abe K. Shimizu T. FEBS Lett. 1992; 309: 353-357Crossref PubMed Scopus (36) Google Scholar). 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 (27Blomster M. Wetterholm A. Mueller M.J. Haeggström J.Z. Eur. J. Biochem. 1995; 231: 528-534Crossref PubMed Scopus (56) Google Scholar). 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 members of the M1 family of metallopeptidases and proposed to play a role in peptide substrate binding (28Barret A.J. Rawlings N.D. Woessner J.F. Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, San Diego1998: 994-996Google Scholar). 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 (29Rudberg P.C. Tholander F. Thunnisen M.G.M. Haeggström J.Z. J. Biol. Chem. 2002; 277: 1398-1404Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). 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 (30Rudberg P.C. Tholander F. Thunnissen M.M. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4215-4220Crossref PubMed Scopus (36) Google Scholar). 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 (31Rudberg P.C. Tholander F. Andberg M. Thunnissen M.M. Haeggström J.Z. J. Biol. Chem. 2004; 279: 27376-27382Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The structure of LTA4H in complex with the competitive inhibitor bestatin has been determined at 1.95-Å resolution (32Thunnissen M.G.M. Nordlund P. Haeggström J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (254) Google Scholar). 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 × 65 × 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 (33Matthews B.W. Fenna R.E. Bolognesi M.C. Schmid M.F. Olson J.M. J. Mol. Biol. 1979; 131: 259-285Crossref PubMed Scopus (214) Google Scholar). 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 (34Groves M.R. Barford D. Curr. Opin. Struct. Biol. 1999; 9: 383-389Crossref PubMed Scopus (279) Google Scholar). 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, and Tyr-383 are located. Behind the pocket occupied by the phenyl ring of bestatin there is an L-shaped hydrophobic cavity ∼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 (35Thunnissen M.M. Andersson B. Samuelsson B. Wong C.-H. Haeggström J.Z. FASEB J. 2002; 16: 1648-1650Crossref PubMed Scopus (65) Google Scholar). 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). The stereochemistry at C-12 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 Δ6-cis-Δ8-trans-Δ10-trans configuration of the conjugated triene. This reaction is unique and requires control of the stereospecific introduction of a hydroxyl group at a site (C-12) distant from the reactive epoxide moiety (C-5—C-6). 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 ensure perfect substrate alignment required for catalysis (31Rudberg P.C. Tholander F. Andberg M. Thunnissen M.M. Haeggström J.Z. J. Biol. Chem. 2004; 279: 27376-27382Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), in agreement with the fact that the free carboxylic acid of LTA4 is required for catalysis (19Ohishi N. Izumi T. Minami M. Kitamura S. Seyama Y. Ohkawa S. Terao S. Yotsumoto H. Takaku F. Shimizu T. J. Biol. Chem. 1987; 262: 10200-10205Abstract Full Text PDF PubMed Google Scholar, 36Maycock A.L. Anderson M.S. DeSousa D.M. Kuehl Jr., F.A. J. Biol. Chem. 1982; 257: 13911-13914Abstract Full Text PDF PubMed Google Scholar). 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 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 of the In this direct a water molecule for attack at C-12 and thus control the and stereospecific of the group in LTB4, in agreement with data (30Rudberg P.C. Tholander F. Thunnissen M.M. Samuelsson B. Haeggström J.Z. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4215-4220Crossref PubMed Scopus (36) Google Scholar). Moreover, the and of the LTA4 binding cavity also suggest the chemical for of the double bond in LTB4. Because there is free between and C-7 of LTA4, the enzyme may this bond in a configuration in the which promote the of a double bond from the carbocation (Fig. 4). The modeled LTA4 molecule then a bent that fits very well with the of the binding the critical double bond geometry at in LTB4 to be by the binding conformation of LTA4 at the active In agreement with has been for thermolysin, the peptide cleaving activity of LTA4H according to a general base B.W. 16: PubMed Scopus Google Scholar). the catalytic zinc is to its three amino acid ligands and an activated water The water is from the zinc by the of the which in turn to the active site via its N-terminal group binding to In this Glu-271 will the and also to the of the enzyme (Fig. 4). The water molecule is by the carboxylate of Glu-296 to promote an attack on the of the peptide the same a proton is to the of the peptide bond by of from and crystallography leads to the that the two enzyme activities of LTA4H are via and active 1 and 4). certain residues are required for the aminopeptidase reaction, i.e. Glu-296 and is critical only for the epoxide hydrolase the other Glu-271, and the zinc are for both In Glu-271 is a unique of a residue that is between two catalytic a in of the two enzyme on its zinc signature and aminopeptidase activity, LTA4H is as a of the M1 family of zinc which enzymes such as aminopeptidase aminopeptidase and aminopeptidase N (28Barret A.J. Rawlings N.D. Woessner J.F. Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, San Diego1998: 994-996Google Scholar). the epoxide hydrolase activity to be unique for LTA4H and has not been with in any human although data have been reported for aminopeptidase S. T. A. A. S. N. P. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, K. M. J. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Google Scholar). LTA4H is in several and but not in J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, F. Haeggström J.Z. FEBS Lett. PubMed Scopus Google Scholar). aminopeptidase 1 from which is at the amino acid to mammalian LTA4H, to LTA4 into LTB4 Ford-Hutchinson A.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the other an LTA4H that is to the human enzyme has been cloned and from F. E. Haeggström J.Z. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The S. LTA4H is a zinc aminopeptidase with a epoxide hydrolase activity against Furthermore, binding of LTA4 to the active site leads to of the epoxide hydrolase activity and activation of the peptidase activity. these data suggest that LTA4H has from an which an lipid binding the was into an active site structural further substrate alignment and catalysis and of LTB4. The specific roles of LTB4 in acute and chronic inflammation have been and by several of biosynthesis or of the (7Byrum R.S. Goulet J.L. Snouwaert J.N. Griffiths R.J. Koller B.H. J. Immunol. 1999; 163: 6810-6819PubMed Google Scholar, C.D. Nature. 1994; PubMed Scopus Google Scholar, N. K. J.A. J. Clin. Invest. 1999; PubMed Scopus Google Scholar, E. T. A. Izumi T. T. S. Shimizu T. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus (80) Google Scholar, B. A R. J. Exp. Med. 2000; 192: PubMed Scopus Google Scholar, A.M. Goodarzi K. Bercury S.D. von U.H. Luster A.D. J. Exp. Med. 2000; 192: PubMed Scopus Google Scholar). this of in vivo data to LTA4H as a for of Moreover, the has been further by the of protein in and host disease cell N. S. N. J. M.J. J. Natl. 2003; PubMed Scopus Google Scholar, T. H. A. E. A. B. H. 2004; PubMed Scopus Google Scholar). and of and also LTA4H L. Gierse J. S. Fitzpatrick F.A. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). In addition, and a known inhibitor of enzymes, are R. A. M. P. N. J. Med. Chem. 1992; PubMed Scopus Google Scholar, R.A. Med. Chem. Lett. 1995; Scopus Google Scholar). Several and have powerful and selective a and a acid were and found to be binding with in the low to nanomolar A. Haeggström J.Z. Samuelsson B. W. B. Wong J. Exp. 1995; Google Scholar, Wetterholm A. Blomster Andberg M. Haeggström J. Samuelsson B. Wong C.-H. Chem. Eur. J. 4: Scopus Google Scholar). of have also been by in particular which LTB4 in human with an of is and arachidonic acid-induced in the J.F. D. N. S. C.D. M.A. J. Exp. 2002; PubMed Scopus Google Scholar, J.F. D. C.D. M.A. J. Exp. 2002; PubMed Scopus Google Scholar). With the structure of LTA4H at it will be to to and selective with