Molecular biology and enzymology of elasmobranch 3β-hydroxysteroid dehydrogenase

Abstract

The conversion of 3b-hydroxy-δ5 steroids into their respective biologically active 3-keto-δ4 steroid counterparts is catalyzed by a microsomal enzyme, 3β-hydroxysteroid dehydrogenase /5-ene-4-ene isomerase (3β-HSD). In order to study this enzyme in elasmobranchs, partial cDNA clones of 3β-HSD were isolated from steroidogenic tissue of the southern stingray (Dasyatis americana), spiny dogfish shark (Squalus acanthias), and the blacktip shark (Carcharhinus limbatus). The deduced amino acid sequences of the elasmobranchs were 40-44% identical to mammalian 3β-HSD sequences, and approximately 50% identical to the rainbow trout (Oncorhynchus mykiss) form. However, the highly conserved substrate-binding region was 80-90% identical among the various species. Northern blot analysis of interrenal gland RNA indicated a single 2.4 kb transcript for the stingray homolog and a 1.4 kb transcript for the blacktip shark homolog. Blacktip shark 3β-HSD activity was enriched in the microsomal fraction of the interrenal gland and was only detected in the presence of the oxidized forms of nicotinamide adenine dinucleotides. Maximal rates were observed near pH 7.5 and between 25 °C and 30 °C. Urea (400 mM) and trimethylamine oxide (200 mM), normally present in elasmobranch blood, had no effect upon 3β-HSD activity. Kinetic parameters of blacktip shark 3β-HSD were determined for pregnenolone (Km = 0.35 ± 0.06 μM; Vmax = 2.3 ± 0.11 nmol min-1 mg protein-1) and DHEA (Km = 0.12 ± 0.02 μM; Vmax = 1.1 ± 0.41 nmol min-1). The kinetic parameters of the stingray 3β-HSD for pregnenolone (Km = 0.13 ± 0.09 μM; Vmax = 4.0 ± 0.51 nmol min-1 mg protein-1) and DHEA (Km = 0.12 ± 0.08 μM; Vmax = 4.1 ± 0.53 nmol min-1 mg protein-1) were similar to those of the blackt