Expression and characterization of the recombinant juvenile hormone epoxide hydrolase (JHEH) from Manduca sexta

• Published in: Insect biochemistry and molecular biology Vol. 28; no. 5; pp. 409 - 419
• Main Authors: Debernard, Stéphane, Morisseau, Christophe, Severson, Tonya F, Feng, Li, Wojtasek, Hubert, Prestwich, Glenn D, Hammock, Bruce D
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.05.1998
• Subjects: Animals; Baculoviridae - genetics; Base Sequence; DNA Primers - genetics; Epoxide Hydrolase; Epoxide Hydrolases - genetics; Epoxide Hydrolases - isolation & purification; Epoxide Hydrolases - metabolism; Gene Expression; Hydrogen-Ion Concentration; Juvenile Hormone; Juvenile Hormones - metabolism; Manduca - enzymology; Manduca - genetics; Manduca sexta; mechanism; Polymerase Chain Reaction; recombinant enzyme; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Manduca sexta; mechanism; recombinant enzyme; Juvenile Hormone; Epoxide Hydrolase
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/S0965-1748(98)00014-9

The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activity). As expected, Ms-JHEH was localized in the microsomal fraction with a molecular mass of approximately 50 kDa. Ms-JHEH showed a substrate and inhibitor spectrum similar to the wild type JHEH isolated from eggs of M. sexta. Its enzymatic activity was the highest for Juvenile Hormone III. Ms-JHEH hydrolyzed several trans-epoxides faster than cis-epoxides. A putative hydroxyl-acyl enzyme intermediate was isolated suggesting a catalytic mechanism of Ms-JHEH similar to the mammalian EHs.