Substrate Report for: HEPTAT

The insect growth regulator (IGR) imidazole KK-42 induces hemolymph juvenile hormone esterase activity and precocious metamorphosis in Bombyx mori. As an initial step to understand the molecular action of KK-42, we isolated a full-length of juvenile hormone esterase cDNA from B. mori (BmJHE). The deduced amino acid sequence of BmJHE showed high identity to JHEs of Heliothis virescens (54%) and Choristoneura fumiferana (52%). Recombinant BmJHE protein expressed in the baculovirus expression system hydrolyzed 3H-JH III and JH analog, HEPTAT, indicating that BmJHE cDNA encodes functional JH esterase. Northern blot analysis showed that the BmJHE transcript was present predominantly in the fat body at the beginning of the last larval instar. During this instar, BmJHE transcript increased gradually until day 7, then decreased, and increased again on day 10 in the fat body. This temporary expression pattern was similar to that of JHE enzyme activity in hemolymph. In contrast, in the 4th instar, the BmJHE transcript was present in the fat body even though hemolymph JHE activity was very low. Western blot analysis using anti-BmJHE antiserum showed BmJHE protein was present in hemolymph during the 5th instar but not during the 4th instar. These results indicate that BmJHE protein is secreted into hemolymph at the metamorphic stage. Hemolymph JHE activity was high in precociously metamorphosed 4th instar larvae (treated KK-42) but low in normal 4th and extra-molted 6th instar larvae (fed 20E). KK-42-treated larvae showed high expression level of BmJHE transcript in the fat body, suggesting that KK-42 enhances BmJHE gene expression in the fat body.

Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.

Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, juvenile hormone esterase (JHE). Substrates were designed that resembled the endogenous substrate juvenile hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman's method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen beta to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylesterase, and acetylcholinesterase were determined for the surrogate substrates. While JHE and porcine liver carboxylesterase hydrolyzed several of the substrates, acetylcholinesterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher Km values but are more easily hydrolyzed (Vmax) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [3H]JH III substrate.

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.

Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. This tight fit places two noncatalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the alpha,beta-unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low K(M) that MsJHE shows for JH. This low K(M), however, comes at the cost of reduced substrate turnover. Neither nucleophilic attack of the resonance-stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appears to be a rate-determining step in the hydrolysis of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism.

Normal insect development requires a precisely timed, precipitous drop in hemolymph juvenile hormone (JH) titer. This drop occurs through a coordinated halt in JH biosynthesis and increase in JH metabolism. In many species, JH esterase (JHE) is critical for metabolism of the resonance-stabilized methyl ester of JH. JHE metabolizes JH with a high kcat/KM ratio that results primarily from an exceptionally low KM. Here we review the biochemistry and structure of authentic and recombinant JHEs from six insect orders, and present updated diagnostic criteria that help to distinguish JHEs from other carboxylesterases. The use of a JHE-encoding gene to improve the insecticidal efficacy of biopesticides is also discussed.

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.

Juvenile hormone regulates the development and reproduction in a variety of insects. Juvenile hormone esterase (JHE) is a selective enzyme, which hydrolyzes the methyl ester of JH and alters its activity. In Tenebrio molitor, JHE has been previously purified from pupae and a partial cDNA was amplified by RT-PCR using fat body mRNA. The previous report indicated that several forms of the JHE protein were present in pupal homogenate. In this study, we report the full-length cDNA, which was obtained by RACE methods. The deduced protein sequence corresponds to peptides from two proteins of different molecular weights in the previous study. The coding region of the full-length cDNA was subcloned into the AcMNPV genome and high levels of expression of the JHE enzyme from the viral p10 promoter were demonstrated in cell culture. The majority of JHE is secreted from the cells as a soluble enzyme. The recombinant JHE enzyme was biochemically characterized. The recombinant protein appears by PAGE analysis as a monomer of approximately the same MW (66000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA.

Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.

The insect growth regulator (IGR) imidazole KK-42 induces hemolymph juvenile hormone esterase activity and precocious metamorphosis in Bombyx mori. As an initial step to understand the molecular action of KK-42, we isolated a full-length of juvenile hormone esterase cDNA from B. mori (BmJHE). The deduced amino acid sequence of BmJHE showed high identity to JHEs of Heliothis virescens (54%) and Choristoneura fumiferana (52%). Recombinant BmJHE protein expressed in the baculovirus expression system hydrolyzed 3H-JH III and JH analog, HEPTAT, indicating that BmJHE cDNA encodes functional JH esterase. Northern blot analysis showed that the BmJHE transcript was present predominantly in the fat body at the beginning of the last larval instar. During this instar, BmJHE transcript increased gradually until day 7, then decreased, and increased again on day 10 in the fat body. This temporary expression pattern was similar to that of JHE enzyme activity in hemolymph. In contrast, in the 4th instar, the BmJHE transcript was present in the fat body even though hemolymph JHE activity was very low. Western blot analysis using anti-BmJHE antiserum showed BmJHE protein was present in hemolymph during the 5th instar but not during the 4th instar. These results indicate that BmJHE protein is secreted into hemolymph at the metamorphic stage. Hemolymph JHE activity was high in precociously metamorphosed 4th instar larvae (treated KK-42) but low in normal 4th and extra-molted 6th instar larvae (fed 20E). KK-42-treated larvae showed high expression level of BmJHE transcript in the fat body, suggesting that KK-42 enhances BmJHE gene expression in the fat body.

Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.

Juvenile hormone esterase (JHE; EC 3.1.1.1), which is intrinsically involved in regulation of development of some insect larvae, is rapidly removed from the hemolymph by the pericardial cells. Lys-29 and Lys-524, which are implicated in the degradation of JHE, were mutated to Arg. Neither the half-life of the modified JHE in the hemolymph nor the catalytic parameters were changed significantly, but when combined, these mutations resulted in apparent failure of lysosomal targeting in the pericardial cell complex. A hypothesis for the mechanism of reduced efficiency of lysosomal targeting is presented. Infection of larvae with a recombinant baculovirus expressing the modified JHE resulted in a 50% reduction in feeding damage compared with larvae infected with the wild-type virus, thus demonstrating improved properties as a biological insecticide. These data demonstrate that alteration of specific residues of JHE that disrupted lysosomal targeting, dramatically increased the insecticidal activity of this protein.

Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, juvenile hormone esterase (JHE). Substrates were designed that resembled the endogenous substrate juvenile hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman's method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen beta to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylesterase, and acetylcholinesterase were determined for the surrogate substrates. While JHE and porcine liver carboxylesterase hydrolyzed several of the substrates, acetylcholinesterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher Km values but are more easily hydrolyzed (Vmax) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [3H]JH III substrate.