Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A₄ epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A₄ (LTA₄) to leukotriene B₄ (LTB₄) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 μM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA₄ to LTB₄. This method was used to produce 700 mg of LTB₄ from LTA₄ methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca²⁺, Mn⁺, Fe²⁺, Zn⁺² and Cu⁺² were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 μM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 μM. From these inhibition studies, it can be theorized that the epoxide hydrolae has at least one hydrophobic and one hydrophilic binding site.     

Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and an apparent Km for leukotriene A4 between 2 X 10(-5) and 3 X 10(-5) M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases.     

Purification and characterization of rat-liver cytosolic epoxide hydrolase

Rat liver cytosolic epoxide hydrolase has been purified and characterized. The enzyme was purified from tiadenol-induced rat liver 540-fold with respect to trans-stilbene oxide as a substrate. Similar purification was obtained with the substrates trans-beta-ethyl styrene oxide and styrene 7,8-oxide, the specific activities decreasing in the order trans-beta-ethyl styrene oxide greater than styrene 7,8-oxide greater than trans-stilbene oxide. The enzyme exerts highest activity at pH 7.4 Km and Vmax of the pure enzyme for trans-stilbene oxide were 1.7 microM and 205 nmol x min-1 x mg protein-1 respectively. With trans-stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol less than methanol less than acetone less than isopropanol = N,N-dimethyl formamide less than acetonitrile less than tetrahydrofuran. The native enzyme, with a molecular mass of 120 kDa, consists of two 61-kDa subunits. Digestion of rat liver cytosolic and microsomal epoxide hydrolase by three proteases resulted in markedly different peptide maps. Western-blot analysis with antiserum against rat liver cytosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofibrate-induced rats. No cross-reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes. A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig, Syrian hamster and New Zealand white rabbit but not with that of green monkey.     

Guinea-pig liver leukotriene A4 hydrolase. Purification, characterization and structural properties

Leukotriene A4 hydrolase from perfused guinea-pig liver was purified 1200-fold to near homogeneity with a yield of about 20%. Apparent values of Km and Vmax at 37 degrees C (27 microM and 68 mumol x mg-1 x min-1), turnover number, and activation energy for the conversion of leukotriene A4 into leukotriene B4 were estimated from kinetic data obtained at -10 degrees C, 0 degree C and +10 degrees C (Arrhenius plots). Physical properties including Mr (67,000-71,000), pH optimum, isoelectric point and Stokes' radius were determined. The amino acid composition and N-terminal amino acid sequence were established after carboxymethylation of the enzyme. Unlike liver cytosolic epoxide hydrolase, the purified enzyme did not catalyze the conversion of leukotriene A4 into (5S,6R)-5,6-dihydroxy-7,9-trans-11,14-cis-icosatetraenoic acid.     

Purification and characterization of a recombinantCaulobacter crescentus epoxide hydrolase

ACaulobacter crescentus epoxide hydrolase (CCEH) from a recombinantEscherichia coli was purified to homogeneity using a three-step procedure. The CCEH protein was purified 7.3-fold with a 22.9% yield in overall activity. The optimal reaction temperature and pH were determined to be 37°C and pH 8.0, respectively. The addition of 10% (v/v) dimethylsulfoxide as a cosolvent improved the enantioselectivity of CCEH for a batch kinetic resolution of racemic indene oxide.     

Purification and characterization of an epoxide hydrolase from the peroxisomal fraction of mouse liver

Epoxide hydrolase (EH) activity has been reported to occur in most subcellular fractions of mouse liver. The EHs in the microsomal and cytosolic fractions have been purified and characterized; however, the nature of the EH(s) in the peroxisomal fraction is not known. Therefore an EH, pEH, was purified from the solubilized 12,000g fraction, which contain peroxisomes. Previous studies have demonstrated that the EH activity in this crude solubilized 12,000g fraction resides mostly in the peroxisomes. Thus the crude 12,000g pellet from mouse liver, free from cytosolic contamination, was sonicated to obtain a 105,000g soluble fraction containing 80% of the original EH activity in this fraction. The pEH was purified, using trans-stilbene oxide (TSO) as substrate, by a combination of affinity and hydroxyapatite chromatography. The purified pEH had a native molecular weight of 57 kDa, a molecular weight of 59 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7. The purified pEH was observed to be immunologically similar to the cytosolic EH (cEH). The kinetics of hydrolysis of TSO, however, were slightly different. Lineweaver-Burk plots for the inhibition of pEH suggest a probable noncompetitive, mixed-type inhibition. The purified pEH thus appears to be very similar to the cEH. There are minor differences between the purified cEH and pEH, particularly in the kinetic parameters. However, these minor differences are insignificant. These results demonstrate that the cEH and pEH are substantially similar, if not identical.     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Purification of an epoxide hydrolase from Rhodotorula glutinis

The epoxide hydrolase from Rhodotorula glutinis was isolated and initially characterized. The enzyme was membrane associated and could be solubilized by Triton X-100. Purification yielded an enzyme with sp. act. of 66 mol 1,2-epoxyhexane hydrolyzed min^–1 mg^–1 protein. The enzyme was not completely purified to homogeneity but, nevertheless, a major protein was isolated by SDS-PAGE for subsequential amino acid determination of peptide fragments. From sequence alignments to related enzymes, a high homology towards the active site sequences of other microsomal epoxide hydrolases was found. Molecular mass determinations indicated that the native enzyme exists as a homodimer, with a subunit molecular mass of about 45 kDa. Based upon these, this epoxide hydrolase is structurally related to other microsomal epoxide hydrolases.     

Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae

In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Purification and characterization of porcine skeletal muscle aminopeptidase T, a novel metallopeptidase homologous to leukotriene A4 hydrolase

A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS-PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A(4) hydrolase (LTA(4)H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.     

Mechanism-based inactivation of leukotriene A4 hydrolase/aminopeptidase by leukotriene A4. Mass spectrometric and kinetic characterization.

"Suicide" inactivation of leukotriene (LT) A4 hydrolase/aminopeptidase occurs via an irreversible mechanism-based process which is saturable, of pseudo firstorder, and dependent upon catalysis. Data obtained with either recombinant enzyme or enzyme purified from human leukocytes were similar. Apparent binding constants and inactivation rate constants are equivalent, compatible with a single type of substrate-enzyme complex which partitions between two fates, turnover and inactivation. Both catalytic functions are inactivated, consistent with an overlapping active site for this bifunctional enzyme. The partition ratio (turnover/inactivation) for the LTA4-enzyme complex is 129 +/- 16 for LTA4 hydrolase activity and 124 +/- 10 for aminopeptidase activity. The pH dependence for turnover and inactivation are indistinguishable with a maximum at pH 8. L-Proline p-nitroanilide, a weak substrate with a high Km for the aminopeptidase affords only partial protection against inactivation by LTA4. However, two potent competitive inhibitors, bestatin and captopril, protect both catalytic processes from inactivation, consistent with an active-site specificity for the suicide event. Electrospray ionization mass spectrometry indicates that the molecular weight of pure recombinant enzyme is 69,399 +/- 4 and that covalent modification accompanies catalysis, producing an LTA4:enzyme adduct with a molecular weight 69,717 +/- 4 and a 1:1 stoichiometry. In agreement with kinetic data, electrospray ionization mass spectrometry shows that bestatin inhibits the covalent modification of enzyme by LTA4 and that the extent of modification is proportional to the loss of enzymatic activity.     

Azabenzthiazole inhibitors of leukotriene A4 hydrolase

Previously, benzthiazole containing LTA₄H inhibitors were discovered that were potent (1–3), but were associated with the potential for a hERG liability. Utilizing medicinal chemistry first principles (e.g., introducing rigidity, lowering c Log D) a new benzthiazole series was designed, congeners of 1–3, which led to compounds 7a, 7c, 12a–d which exhibited LTA₄H IC₅₀ = 3–6 nM and hERG Dofetilide Binding IC₅₀ = 8.9–> >10 μM.     

Leukotriene A4 hydrolase: an epoxide hydrolase with peptidase activity

Purified leukotriene A4 hydrolase from human leukocytes is shown to exhibit peptidase activity towards the synthetic substrates alanine-4-nitroanilide and leucine-4-nitroanilide. The enzymatic activity is abolished after heat treatment (70 degrees C, 30 min). At 37 degrees C these substrates are hydrolyzed at a rate of 380 and 130 nmol/mg/min, respectively, and there is no enzyme inhibition during catalysis. Apo-leukotriene A4 hydrolase, obtained by removal of the intrinsic zinc atom, exhibits only a low peptidase activity which can be restored by the addition of stoichiometric amounts of zinc. Reconstitution of the apoenzyme with cobalt results in a peptidase activity which exceeds that of enzyme reactivated with zinc. Preincubation of the native enzyme with leukotriene A4 reduces the peptidase activity. Semipurified preparations of bovine intestinal aminopeptidase and porcine kidney aminopeptidase do not hydrolyze leukotriene A4 into leukotriene B4.     

Structure and catalytic mechanisms of leukotriene A4 hydrolase

Leukotriene A4 hydrolase catalyzes the final and committed step in the biosynthesis of leukotriene B4, a potent chemotactic agent for neutrophils, eosinophils, monocytes, and T-cells that play key roles in the innate immune response. Recent data strongly implicates leukotriene B4 in the pathogenesis of cardiovascular diseases, in particular arteriosclerosis, myocardial infarction and stroke. Here, we highlight the most salient features of leukotriene A4 hydrolase with emphasis on its biochemistry and structure biology.     

Cloning and characterization of a microsomal epoxide hydrolase from Heliothis virescens

Epoxide hydrolases (EHs) are α/β-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs). In this study we obtained a full-length cDNA, hvmeh1, from the generalist feeder Heliothis virescens that encoded a highly active EH, Hv-mEH1. Of the 10 different EH substrates that were tested, Hv-mEH1 showed the highest specific activity (1180 nmol min^?1 mg^?1) for a 1,2-disubstituted epoxide-containing fluorescent substrate. This specific activity was more than 25- and 3900-fold higher than that for the general EH substrates cis-stilbene oxide and trans-stilbene oxide, respectively. Although phylogenetic analysis placed Hv-mEH1 in a clade with some lepidopteran JH metabolizing EHs (JHEHs), JH III was a relatively poor substrate for Hv-mEH1. Hv-mEH1 showed a unique substrate selectivity profile for the substrates tested in comparison to those of MsJHEH, a well-characterized JHEH from Manduca sexta, and hmEH, a human microsomal EH. Hv-mEH1 also showed unique enzyme inhibition profiles to JH-like urea, JH-like secondary amide, JH-like primary amide, and non-JH-like primary amide compounds in comparison to MsJHEH and hmEH. Although Hv-mEH1 is capable of metabolizing JH III, our findings suggest that this enzymatic activity does not play a significant role in the metabolism of JH in the caterpillar. The ability of Hv-mEH1 to rapidly hydrolyze 1,2-disubstituted epoxides suggests that it may play roles in the metabolism of fatty acid epoxides such as those that are commonly found in the diet of Heliothis.     

Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34

A putative epoxide hydrolase-encoding gene was identified from the genome sequence of Cupriavidus metallidurans CH34. The gene was cloned and overexpressed in Escherichia coli with His(6)-tag at its N-terminus. The epoxide hydrolase (CMEH) was purified to near homogeneity and was found to be a homodimer, with subunit molecular weight of 36 kDa. The CMEH had broad substrate specificity as it could hydrolyze 13 epoxides, out of 15 substrates tested. CMEH had high specific activity with 1,2-epoxyoctane, 1,2-epoxyhexane, styrene oxide (SO) and was also found to be active with meso-epoxides. The enzyme had optimum pH and temperature of 7.5 and 37°C respectively, with racemic SO. Biotransformation of 80 mM SO with recombinant whole E. coli cells expressing CMEH led to 56% ee(P) of (R)-diol with 77.23% conversion in 30 min. The enzyme could hydrolyze (R)-SO, ～2-fold faster than (S)-SO, though it accepted both (R)- and (S)-SO with similar affinity as K(m)(R) and K(m)(S) of CMEH were 2.05±0.42 and 2.11±0.16 mM, respectively. However, the k(cat)(R) and k(cat)(S) for the two enantiomers of SO were 4.80 and 3.34 s(-1), respectively. The wide substrate spectrum exhibited by CMEH combined with the fast conversion rate makes it a robust biocatalyst for industrial use. Regioselectivity studies with enantiopure (R)- and (S)-SO revealed that with slightly altered regioselectivity, CMEH has a high potential to synthesize an enantiopure (R)-PED, through an enantioconvergent hydrolytic process.     

Thermodynamic properties of leukotriene A4 hydrolase inhibitors

The leukotriene A₄ hydrolase (LTA₄H) is a bifunctional enzyme, containing a peptidase and a hydrolase activity both activities having opposing functions regulating inflammatory response. The hydrolase activity is responsible for the conversion of leukotriene A₄ to pro-inflammatory leukotriene B₄, and hence, selective inhibitors of the hydrolase activity are of high pharmacological interest. Here we present the thermodynamic characterization of structurally distinct inhibitors of the LTA₄H that occupy different regions of the binding site using different biophysical methods. An in silico method for the determination of stabilized water molecules in the binding site of the apo structure of LTA₄H is used to interpret the measured thermodynamic data and provided insights for design of novel LTA₄H inhibitors.     

菜豆环氧化物水解酶的表达及其催化特性研究

【背景】光学纯环氧化物及邻二醇是一类多功能手性砌块。与化学合成法相比,环氧化物水解酶(EHs)介导的生物转化法因环境友好而成为当前的研究热点。【目的】从菜豆(Phaseolus vulgaris)中克隆一种EH基因并进行原核表达,研究重组酶催化环氧苯乙烷(Styrene oxide,SO)的水解特性。【方法】通过计算机辅助分析EHs的一级结构,推测一种菜豆来源的未知功能蛋白(PvEH4)可能具有EH活性。利用RT-PCR技术,以菜豆总RNA为模板,扩增编码PvEH4的基因pveh4,并实现其在Escherichia coli BL21(DE3)中的表达。利用重组菌E.coli/pveh4全细胞催化SO水解,分析PvEH4的对映选择性和区域选择性。【结果】一级结构分析表明,PvEH4具有α/β折叠型EH特有的保守区域。SDS-PAGE结果显示PvEH4的表观分子量为39.4 kD。PvEH4针对SO的对映选择率(E值)为10.1,区域选择性系数αS和βR分别为99.5%和82.5%。当外消旋SO转化率达到68.1%时,可同时获得99.9%ees的(R)-SO和92.3%eep的(R)-苯乙二醇(Phenyl-1,2-ethanediol,PED),两者的产率分别为31.9%和65.6%。【结论】PvEH4的挖掘不仅增加了植物类EHs的数量,同时也为EHs的蛋白分子改造提供参考。