Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis

Ward, Vernon K.; Bonning, Bryony C.; Huang, Tien L.; Shiotsuki, Takahiro; Griffeth, Valerie; Hammock, Bruce D.

doi:10.1016/0020-711x(92)90289-d

Cited by 66 publications

(56 citation statements)

References 19 publications

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“…A virus expressing modified JHE altered by site-directed mutagenesis of the active site serine to produce inactive enzyme, was used as a negative control. This virus is identical to AcUW2(B).JHE except that the protein expressed has no catalytic activity (Ward et al, 1992). The expression characteristics of a recombinant virus employing the basic protein promoter were also analysed in this study.…”

Section: Methodsmentioning

confidence: 99%

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters

Bonning

Roelvink

Vlak

et al. 1994

Journal of General Virology

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The expression characteristics of the pl0, polyhedrin and basic protein promoters of Autographa californica nuclear polyhedrosis virus were compared using two reporter enzymes, juvenile hormone esterase (JHE) and fl-galactosidase. In these systems, JHE is exported from the cell and fl-galactosidase is localized to the cytosol. Expression of JHE from the basic, pl0 and polyhedrin promoters was first detected in the medium at 13, 19 and 27 h post-infection respectively. The basic protein promoter yielded the highest expression of the three promoters tested for both enzymes, as determined by protein and enzyme activity assays. In addition, yields of fl-galactosidase and JHE under control of the pl0 promoter are higher relative to expression under control of the polyhedrin promoter. These data highlight the importance of investigation of viral promoters other than the polyhedrin promoter for high yield protein expression in vitro, and for insecticidal use of recombinant baculoviruses requiring high levels of expression. The results support revision of the current concept that very late viral promoters are always optimal for high yield recombinant protein expression.

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Section: Methodsmentioning

confidence: 99%

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters

Bonning

Roelvink

Vlak

et al. 1994

Journal of General Virology

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“…The catalytic triad of JHE is predicted to be composed of Ser 203 -His 448 -Glu 334 based on site-directed mutagenesis and amino acid homology. 11 Of the X-ray crystal structures known to be members of the ␣/␤ hydrolase fold family, JHE displays the highest se-quence homology to acetylcholinesterase. Interestingly, both JHE and acetylcholinesterase have been established as key regulatory enzymes that are involved in the catabolism of chemical mediators, juvenile hormone, and acetylcholine, respectively.…”

Section: Introductionmentioning

confidence: 99%

Homology model of Juvenile Hormone Esterase from the crop pest,Heliothis virescens

et al. 1999

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Juvenile Hormone Esterase (JHE) plays an essential role in the development of insects since it is partially responsible for clearing juvenile hormone (JH), one of the hormones that is responsible for insect metamorphosis. JHE is a 60 kDa enzyme that selectively hydrolyzes the ␣/␤ unsaturated ester of JH. Because of its pivotal role in insect development, we have targeted JHE for use as a biopesticide. In this study, we have constructed a homology-based molecular model of JHE from the agricultural crop pest, Heliothis virescens. JHE is a member of the ␣/␤ hydrolase fold family of enzymes and was built according to two structures in the same family: acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum. Analysis of the active site region reveals extensive conservation between JHE and its templates. A surprise was the presence of a conserved Ser near the catalytic triad. Docking of JH III into the active site has provided insight into protein-substrate interactions that are corroborated by experimental observation. The model is being used as a predictive basis to design biopesticides. In this regard, we have identified a site on the protein surface that is suggestive of a recognition site for the putative JHE receptor. Proteins 1999; 34:184-196.1999 Wiley-Liss, Inc.

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“…When NSJHE were produced on a large scale, HIGH FIVE cells were infected with 10 plaque forming units per cell and cultured in spinner flasks. An original recombinant virus (AcJHE) producing a secreted form of JHE (rJHE) 12 was used as a positive control and AcMNPV-C6 was used as a negative control for the enzyme assay, ELISA and Western blots.…”

Section: Cells and Virus Infectionmentioning

confidence: 99%

Expression of a Non-Secreted Form of Juvenile Hormone Esterase in a Baculovirus

Taniai

Zhou

Lee

et al. 2005

JARQ

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Pericardial cells rapidly cleared recombinant-juvenile hormone esterase (rJHE) expressed within a baculovirus in insects from the hemolymph. To prevent the clearance of rJHE, we used the polymerase chain reaction (PCR) to remove the signal sequence from the JHE gene, thereby converting the enzyme to a non-secreted form (NSJHE). The resulting gene was expressed in a baculovirus (AcN-SJHE) using HIGH-FIVE cells and proper cellular enzyme production was monitored. Transcription level of the NSJHE and rJHE were comparable, and purified NSJHE protein from the cytoplasm hydrolyzed JH. However, efficacy of NSJHE production was low. Enzyme-linked immunosorbent assays and enzyme assays demonstrated enzyme production and activity relative to rJHE of 0.5-1.7% and < 0.1%, respectively. Transmission electron microscopy (TEM) revealed that NSJHE was distributed in the nucleus predominantly and some NSJHE aggregated in clumps within the cytoplasm. These results indicate that NSJHE lacks a specific localization site within cells and that the folding of this enzyme is insufficient. Deglycosylation experiments using purified NSJHE showed that NSJHE was much less glycosylated than rJHE as we expected. Although the NSJHE was less glycosylated, enzyme stability of the NSJHE was equivalent to that of rJHE, indicating that the sugar chains are unimportant in the stability of JHE.

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Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis

Cited by 66 publications

References 19 publications

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters

Homology model of Juvenile Hormone Esterase from the crop pest,Heliothis virescens

Expression of a Non-Secreted Form of Juvenile Hormone Esterase in a Baculovirus

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