Abstract
Ecdysteroids are critically important for the formation of the insect exoskeleton. Cholesterol is a precursor of ecdysone and its active form 20-hydroxyecdysone, but some steps in the ecdysteroid biosynthesis pathway remain unknown. An essential requirement of glutathione (GSH) transferase GSTE14 in ecdysteroid biosynthesis has been established in Drosophilamelanogaster, but its function is entirely unknown. Here, we have determined the crystal structure of GSTE14 in complex with GSH and investigated the kinetic properties of GSTE14 with alternative substrates. GSTE14 has high-ranking steroid double-bond isomerase activity, albeit 50-fold lower than the most efficient mammalian GSTs. Corresponding steroid isomerizations are unknown in insects, and their exact physiological role remains to be shown. Nonetheless, the essential enzyme GSTE14 is here demonstrated to be catalytically competent and have a steroid-binding site.
Highlights
FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies
Certain members in the family of glutathione transferases (GSTs), enzymes originally discovered as detoxication enzymes [1], have been shown to fulfill significant functions in steroid hormone biosynthesis
Loss of the GSTE14 gene is detrimental to cuticle formation, it interrupts the formation of exoskeleton and prevents ecdysis, but the effect can be rescued by the administration of 20-hydroxyecdysone
Summary
DNA encoding the D. melanogaster GSTE14 (DmGSTE14) protein sequence (NP_610855.1) was codon-optimized for Escherichia coli expression and designed to include a His6tag at the C terminus. The 50 mL culture was used to inoculate 4 L LB-ampicillin medium. Cells were harvested by centrifugation (4000 g, 20 min, 4 °C), resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 100 lM DTT, and 20 mM imidazole, pH 7.4), and disrupted at 4 °C using sonication (Vibracell; Bioblock, Waltham, MA, USA). Following washing with binding buffer, DmGSTE14 was eluted using 20 mM sodium phosphate, 0.5 M NaCl, 100 lM DTT, and 500 mM imidazole, pH 7.4. DmGSTE14 was concentrated using an Amicon ultra-spin column with a cutoff of 10 kDa (Millipore, Billerica, MA, USA). Protein purity was confirmed using a Coomassiestained 12% SDS/PAGE
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