Allosteric interaction between 3β‐hydroxysteroid dehydrogenase/Δ 5 ‐Δ 4 isomerase and cytochrome b 5 influences cofactor binding

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 + .—Goosen, P., Swart, A. C., Storbeck, K.‐H., Swart, P. Allosteric interaction between 3β‐hydroxysteroid dehydrogenase/Δ 5 ‐Δ 4 isomerase and cytochrome b 5 influences cofactor binding. FASEB J. 27, 322–332 (2013). www.fasebj.org