title: Leukotriene A4 hydrolase: selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375.
abstract: Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) hydrolase (LTA4H)/aminopeptidase is a bifunctional zinc metalloenzyme that catalyzes the final and rate-limiting step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattractant and immune modulating lipid mediator. Two chemical features are key to the bioactivity of LTB4, namely, the chirality of the 12R-hydroxyl group and the cis-trans-trans geometry of the conjugated triene structure. From the crystal structure of LTA4H, a hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was identified in a narrow and otherwise hydrophobic pocket, believed to bind LTA4. In addition, Asp-375 belongs to peptide K21, a previously characterized 21-residue active site-peptide to which LTA4 binds during suicide inactivation. In the present report we used site-directed mutagenesis and x-ray crystallography to show that Asp-375, but none of the other candidate residues, is specifically required for the epoxide hydrolase activity of LTA4H. Thus, mutation of Asp-375 leads to a selective loss of the enzyme's ability to generate LTB4 whereas the aminopeptidase activity is preserved. We propose that Asp-375, possibly assisted by Gln-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Rudberg
authors.forename: Fredrik
authors.initials: F
authors.lastname: Tholander
authors.forename: Marjolein M G M
authors.initials: MM
authors.lastname: Thunnissen
authors.forename: Bengt
authors.initials: B
authors.lastname: Samuelsson
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggstrom
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid; D020134:Catalytic Domain; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D007975:Leukotriene B4 / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D007975:Leukotriene B4; D001224:Aspartic Acid; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase