The biosynthesis of steroid hormones, essential to the survival of all mammals, is dependent on the activity of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD). 3βHSD activity is, in turn, influenced by cytochrome-b(5) (Cyt-b(5)). However, the mechanism through which this occurs is unknown. In this study, we investigated this mechanism by evaluating the influence of Cyt-b(5) on the dehydrogenase and isomerase activities of 3βHSD. Capra hircus 3βHSD was overexpressed in SF-9 cells, using a baculovirus expression system, and purified. Substrate and cofactor kinetics were determined spectrophotometrically in the presence and absence of purified Ovis aries liver Cyt-b(5). Nonspecific enzyme activity was evaluated by zero-enzyme, -substrate, and -cofactor blanks. Fusion proteins, 3βHSD-eCFP, and Cyt-b(5)-eYFP were subsequently coexpressed in COS-1 cells and analyzed for FRET. A CFP-YFP fusion protein served as positive control, while coexpression of 3βHSD-eCFP and cytochrome P450 17α-hydroxylase/17,20 lyase-eYFP (CYP17A1-eYFP) served as negative control. Results showed Cyt-b(5) to decrease the K(m,)(NAD(+)) value of 3βHSD ≈3.5-fold while increasing the V(max,app) of the dehydrogenase reaction ≈17. FRET analysis showed COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b(5)-eYFP to exhibit a FRET signal ≈9-fold greater than that of the negative control. These results indicate that Cyt-b(5) augments 3βHSD activity via an allosteric mechanism by increasing the affinity of the enzyme toward NAD(+).
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