Saccharomyces cerevisiae leukotriene A(4) hydrolase: Formation of leukotriene B-4 and identification of catalytic residues

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B-4, and also possesses an aminopeptidase activity. Re cently we cloned and characterized an leukotriene A(4) hydrolase from Sacch aromyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase ac tivity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a met alloenzyme containing one zinc atom complexed to His-340, His-344, and Glu- 363. Mutagenetic analysis indicates that the aminopeptidase activity follow s a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A (4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosa tetraenoic acid, leukotriene B-4, and Delta (6)-trans-Delta (8)-cis-leukotr iene B-4, with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively in activates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and m utated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A( 4) binds in one catalytic mode and one tight-binding, regulatory mode. Exch ange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, th e corresponding residue in human leukotriene A(4) hydrolase, results in a p rotein that converts leukotriene A(4) into leukotriene B-4 with an improved efficiency and specificity. Hence, by a single point mutation, we could ma ke the active site better suited to bind and turn over the substrate leukot riene. A(4), thus mimicking a distinct step in the molecular evolution of S . cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.