Evolution and structure of leukotriene A4 hydrolase

Sammanfattning: Leukotriene (LT) B4 is a potent leukocyte chemotactic and aggregating agent involved in a variety of inflammatory and allergic disorders. The enzyme leukotriene A4 hydrolase catalyzes the hydrolysis of LTA4 into this proinflammatory mediator, leukotriene B4. The mammalian LTA4 hydrolase is a zinc metalloenzyme (69 kDa) that also possesses an anion-dependent peptidase activity, for which the physiological role is yet unknown. The zinc atom is required for both enzymatic activities and is bound to His-295, His-299 and Glu-318, components of the zinc binding motif. During catalysis the enzyme is suicide inactivated by the substrate LTA4, both enzyme activities are abolished. LTA4 hydrolase is homologous to other zinc aminopeptidases in a variety of species, in particular the enzymes belonging to the M1 family. The epoxide hydrolase activity is not as widespread among members of this family. Formation of LTB4 has been detected in vertebrates, but not in lower species. To gain information about the evolution of LTA4 hydrolase and its two catalytic activities, we have studied LTA4 hydrolase in the toad, Xenopus laevis, and yeast, Saccharomyces cerevisiae. LTA4 hydrolase in X laevis has a wide organ distribution with high levels in the intestine and the reproduction organs. Purification and characterization revealed a zinc metalloenzyme (69 kDa) with an anion-dependent peptidase activity. The toad LTA4 hydrolase converts LTA4 into LTB4 and delta6-trans-delta8-cis-LTB4, a metabolite which contracts the guinea pig lung parenchyma. The catalytic efficiency of X. laevis LTA4 hydrolase is higher as compared to the human enzyme. The specificity constants indicated that the active site is equally well adapted to accomodate LTA4 and the peptidase substrate, alanine-p-nitroanilide, as compared to the human LTA4 hydrolase. Western and Northern blot analysis revealed structural differences between the X. laevis and the human LTA4 hydrolase, and amino acid sequencing of internal peptides of the toad enzyme showed that it is 60% identical with the human enzyme. S. cerevisiae LTA4 hydrolase was cloned and characterized as a zinc containing anionactivated leucyl aminopeptidase with a molecular mass of 72 kDa. By site directed mutagenesis the zinc atom was shown to be bound to His-340, His-344 and Glu-363. The yeast enzyme was able to hydrolyze LTA4 into 5S,6S-DHETE, LTB4 and delta6-trans-delta8-cis-LTB4. Mutagenetic analysis suggested that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Exposure of S. cerevisiae LTA4 hydrolase to LTA4 selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. The binding of LTA4 was shown to be tight and located at the active center of the enzyme. Exchange of Tyr-429 for a Phe partly protected the protein from LTA4 inactivation. By a single point mutation, Phe-424 into a Tyr, the active site became better suited to bind and turn over LTA4, thus mimicking a distinct step in the molecular evolution of S. cerevisiae LTA4 hydrolase towards its mammalian counterparts. Homology modelling, using the crystal structure of human LTA4 hydrolase as template, suggested that the hydrophobic pocket of S. cerevisiae LTA4 hydrolase is wider and shallower. To make the hydrophobic pocket deeper, Gln-412 was converted into a Val, the corresponding residue in human LTA4 hydrolase. This mutation increased the epoxide hydrolase activity. Furthermore, Glu-316 was shown to be a critical residue in both catalytic reactions. These data indicate that evolution has not introduced any new functional residues in the active site to accomplish epoxide hydrolase activity. It seems more likely that subtle structural rearrangements of the active site of a zinc aminopeptidase has occurred to allow use of already existing residues for the second enzyme activity.