Leukotriene A4 hydrolase:: Selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375

Leukotriene A(4) (LTA(4), 5S-traps-5,6-oxido-7,9-traps-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 B-4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-traps-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 L.TA4H. 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 Gin-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4.