Substrate Report for: Juvenile-hormone-III

Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural substrates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible development of novel drugs for human diseases.

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.

Juvenile hormone esterase (JHE) from the prepupal stage of Drosophila melanogaster was purified about 429-fold to near homogeneity by selective precipitations, isoelectric focussing, anion exchange and gel filtration chromatography. The KM and Vmax of the purified enzyme for juvenile hormone III (JHIII) hydrolysis are 89 nM and at least 590 nmol/min/mg, respectively. JHE also hydrolyses the artificial substrate alpha-naphthyl acetate with a KM of 120 micro M and a Vmax of at least 70 mumol/min/mg. Competition of JHIII hydrolysis by five juvenile hormones and twenty-four JH analogues showed JHE is highly selective for JHIII and JHIII bisepoxide (JHP3), and both may be in vivo substrates. Binding in the active site of JHE is promoted by structural features found in JHIII and JHB3 including the epoxide groups in their natural orientations, methyl (rather than ethyl) side-chains, and the 2E, 3 double bond that is conjugated with the ester group. Binding is reduced by almost any departure from these structural features of JH. Co-incubation of the haemolymph JH binding protein, lipophorin, with JHE indicates lipophorin might modulate JH hydrolysis by competition for binding of JH.

Aspongopus chinensis is a Chinese traditional edible and medicinal insect, which is in great demand in the society. This insect reproduces once a year which is caused by reproductive diapause resulting in insufficient production in wild resources. However, the mechanism of diapause in A. chinensis is still unclear. In this study, we focus on the relationship between juvenile hormones (JHs) and A. chinensis diapause. The results showed that JHIII concentration in diapause adult individuals was significantly lower than that in diapause termination adult individuals. When exogenous JHIII was injected into diapause adults, the rate of mating was increased significantly, development of the reproductive systems was accelerated, consumption of fat intensified, the expression of juvenile hormone acid o-methyl-transferase (JHAMT) was upregulated, and juvenile hormone epoxide hydrolase (JHEH) and fatty acid synthase (FAS) gene expressions were downregulated. In addition, RNAi of JHAMT decreased JH concentration, delayed the development of reproductive systems, slowed down fat consumption, and delayed the mean mating occurrence time significantly. Conversely, RNAi of JHEH resulted in an increased concentration of JH, development of reproductive systems was accelerated, consumption of fat was intensified, and mean mating occurrence time advanced significantly. Taken together, these findings uncovered that JH plays an important role in regulating reproductive diapause in A. chinensis and, thus, could provide a theoretical basis for further research on the diapause of A. chinensis.

Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural substrates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible development of novel drugs for human diseases.

In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35 kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects.

In order to understand the roles of the epoxide hydrolases (EHs) in xenobiotic biotransformation in insects, we examined the induction of EHs by exogenous compounds in Drosophila melanogaster third instar larvae. Among the chemicals tested, clofibrate, a phenoxyacetate hypolipidermics drug, increased EH activity towards cis-stilbene oxide approximately twofold in larval whole-body homogenates. The same dose of clofibrate also induced glutathione S-transferase activity. The effect of clofibrate on EH induction was dose-dependent and the highest activity occurred with a 10% clofibrate application. Three other substrates conventionally used in EH assays (trans-stilbene oxide, trans-diphenylpropene oxide and juvenile hormone III) were poorly hydrolysed by larval homogenates, with or without clofibrate administration. Because the increased EH activity was localized predominantly in the microsomal fraction, we synthesized degenerate oligonucleotide primers with sequences corresponding to conserved regions of known microsome EHs from mammals and insects in order to isolate the gene. The 1597 bp putative cDNA of D. melanogaster microsomal EH (DmEH) obtained from a larval cDNA library encoded 463 amino acids in an open reading frame. Northern blot analysis showed that the transcription of DmEH was increased in larvae within 5 h of clofibrate treatment. Recombinant DmEH expressed in baculovirus hydrolysed cis-stilbene oxide (23 nmol.min-1.mg protein-1) and was located mainly in the microsomal fraction of virus-infected Sf9 cells. There was no detectable EH activity toward juvenile hormone III. These observations suggest that DmEH is involved in xenobiotic biotransformation, but not in juvenile hormone metabolism, in D. melanogaster.

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.

A full-length cDNA encoding for a microsomal juvenile hormone (JH)-metabolizing epoxide hydrolase (TmEH-1) was isolated from a cDNA library constructed from fat body of last stadium (wandering) cabbage loopers, Trichoplusia ni, at the exact developmental time of maximum JH epoxide hydrolase activity. TmEH-1 was 1887 base pairs in length with a 1389 base pair open reading frame encoding 463 amino acids. Amino acid sequence analysis showed that TmEH-1 was most similar to and contained the exact catalytic triad (Asp-226, Glu-403 and His-430) found in microsomal epoxide hydrolases. TmEH-1-specific message was present along with JH III epoxide hydrolase activity in fat body in feeding (days 1 and 2) and wandering (day 3) larvae with the peak in message level preceding the peak in JH epoxide hydrolase activity by 1 day. When TmEH-1 was expressed in baculovirus-infected Spodoptera frugiperda cells, a 46,000 molecular weight protein appeared on SDS-PAGE which corresponded to the predicted size coded by the TmEH-1 message and which was positively correlated with increases in JH III epoxide hydrolase activity above that of wild-type controls. In subcellular distribution studies, 58% of the juvenile hormone III epoxide hydrolase activity was in the insoluble fractions. Baculovirus expressed TmEH-1 demonstrated a higher specific activity for JH III as compared to the general EH substrates, cis- and trans-stilbene oxide. Southern blot analyses suggested that multiple epoxide hydrolase genes are present in T. ni.

Juvenile hormone esterase (JHE) from the prepupal stage of Drosophila melanogaster was purified about 429-fold to near homogeneity by selective precipitations, isoelectric focussing, anion exchange and gel filtration chromatography. The KM and Vmax of the purified enzyme for juvenile hormone III (JHIII) hydrolysis are 89 nM and at least 590 nmol/min/mg, respectively. JHE also hydrolyses the artificial substrate alpha-naphthyl acetate with a KM of 120 micro M and a Vmax of at least 70 mumol/min/mg. Competition of JHIII hydrolysis by five juvenile hormones and twenty-four JH analogues showed JHE is highly selective for JHIII and JHIII bisepoxide (JHP3), and both may be in vivo substrates. Binding in the active site of JHE is promoted by structural features found in JHIII and JHB3 including the epoxide groups in their natural orientations, methyl (rather than ethyl) side-chains, and the 2E, 3 double bond that is conjugated with the ester group. Binding is reduced by almost any departure from these structural features of JH. Co-incubation of the haemolymph JH binding protein, lipophorin, with JHE indicates lipophorin might modulate JH hydrolysis by competition for binding of JH.

In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.