title: Allosteric interaction between 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase and cytochrome b5 influences cofactor binding.
abstract: 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(+).
authors.affiliation: Department of Biochemistry, University of Stellenbosch, Stellenbosch 7602, South Africa.
authors.forename: Pierre
authors.initials: P
authors.lastname: Goosen
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Swart
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Storbeck
authors.forename: Pieter
authors.initials: P
authors.lastname: Swart
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D001483:Base Sequence; D015786:Cytochromes b5 / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D013251:Steroid Isomerases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D015786:Cytochromes b5; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D013251:Steroid Isomerases