Search

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr(383) and Tyr(466)) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO(-)) or the ONOO(-) generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO(-) and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr(383) and Tyr(466). Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.

Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.

Endothelial cells contain leukotriene (LT) A4 hydrolase (LTA-H) as detected by Northern and Western blotting, but several studies have been unable to detect the activity of this enzyme. Since LTA-H could play a key role in determining what biologically active lipids are generated by activated endothelium during the inflammatory process, we studied possible mechanisms by which this enzyme may be regulated. We find that LTA-H is phosphorylated under basal conditions in human endothelial cells and in this state does not exhibit epoxide hydrolase activity (i.e. conversion of LTA4 to LTB4). LTA-H purified from endothelial cells is efficiently dephosphorylated by incubation with protein phosphatase-1 in the presence of an LTA-H peptide substrate and not at all in the absence of substrate. Under conditions that lead to dephosphorylation, protein phosphatase-1 activates the epoxide hydrolase activity of LTA-H. Using peptide mapping and site-directed mutagenesis, we have identified serine 415 as the site of phosphorylation of LTA-H by a kinase found in endothelial cell cytosol. In parallel, we have studied a human lung carcinoma cell line that expresses active LTA-H. Although these cells have cytosolic kinases that phosphorylate recombinant LTA-H, they do not target serine 415 and thus do not inhibit LTA-H activity. We believe that LTA-H is regulated in intact cells by a kinase/phosphatase cycle and further that the kinase in endothelial cells specifically recognizes and phosphorylates a regulatory site in the LTA-H.

On the basis of the sequence similarity between mammalian epoxide hydrolases and bacterial haloalkane dehalogenase reported earlier (Arand, M., Grant, D. F., Beetham, J. K., Friedberg, T., Oesch, F., and Hammock, B. D. (1994) FEBS Lett. 338, 251-256; Beetham, J. K., Grant, D., Arand, M., Garbarino, J., Kiyosue, T., Pinot, F., Oesch, F., Belknap, W. R., Shinozaki, K., and hammock, B. D. (1995) DNA Cell. Biol. 14, 61-71) we selected candidate amino acid residues for the putative catalytic triad of the rat soluble epoxide hydrolase. The predicted amino acid residues were exchanged by site-directed mutagenesis of the epoxide hydrolase cDNA, followed by the expression of the respective mutant enzymes in Escherichia coli. A total of 25 different mutants were analyzed for their epoxide hydrolase activity toward the model substrate trans-stilbene oxide. In case of impaired catalytic activity of a given mutant, the structural integrity of the recombinant enzyme protein was assessed either by its ability to covalently bind the substrate trans-stilbene oxide or by affinity purification on benzyl thio-Sepharose, using the soluble epoxide hydrolase-specific competitive inhibitor 4-fluorochalcone oxide to release the bound enzyme from the affinity matrix. Of the mutants under investigation, only those with changes in the positions Asp333, Asp495, and His523 were completely inactive toward the model substrate trans-stilbene oxide while retaining the proper protein fold. These amino acids were exactly those previously predicted by sequence alignment. Exchange of the amino acid residues flanking the catalytic nucleophile Asp333 significantly changed the kinetic properties of the enzyme. Mutation of His332 to Gln had no apparent effect on the Km but led to a heavily reduced Vmax (5% that of the wild type) of the mutant enzyme, while the exchange of Trp334 against Phe strongly increased the Km (7-fold) and also moderately enhanced the Vmax (2-fold) of the corresponding mutant. Mutation of Trp540 apparently had a strong effect on the protein conformation.

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.

Hepatic microsomal epoxide hydrolase activity (EC 3.3.2.3), assayed using styrene oxide as the substrate, has a pH optimum of 9.5 from C57BL/6J mice and a pH optimum of 8.7 from DBA/2J mice. In cross and back-cross matings between C57BL/6J and DBA/2J mice, this phenotypic difference in epoxide hydrolase activity is inherited as a single autosomal trait, with co-dominant expression in heterozygotes. Heating liver microsomes from C57Bl/6J mice at 62 degrees C for 30 min produced a slight decrease in enzyme activity, whereas the same treatment of DBA/2J microsomes reduced enzyme activity to less than 3% of its initial value. Twenty-six inbred strains of mice were examined and separated into two phenotypic classes on the basis of differences in pH optima and heat sensitivity of microsomal epoxide hyrolase activity. Eph-1 is proposed as the locus symbol of the structural gene for microsomal epoxide hydrolase, with superscripts b and d designating the alleles carried by C57BL/6J and DBA/2J mice, respectively. Using 24 recombinant inbred strains derived from C57BL/6J and DBA/2J mice, Eph-1 was found to be linked to two loci on Chromosome 1.