Δ⁵-3β-Hydroxysteroid Dehydrogenase (3βHSD) from Digitalis lanata. Heterologous Expression and Characterisation of the Recombinant Enzyme

During the biosynthesis of cardiac glycosides, Delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3 beta-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 30HSD gene was isolated from leaves of Digitalis lanata. it consisted of 870 nucleotides containing a 90 nucleotide long intron. A full-length cDNA clone that encodes 3 beta HSD was isolated by RT-PCR from the same source. A Sph 1/Kpn 1 3 beta HSD cDNA was cloned into the pQE30 vector and then transferred into E. coli strain M15[pREP4]. 3 beta HSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3 beta HSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). pQ3 beta HSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3 beta-hydroxypregnanes but not cholesterol were 3 beta-oxidised by pQ3 beta HSD when NAD was used as the co-substrate. Testosterone (4-androsten-17 beta-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3 beta HSD has also 17 beta-dehydrogenase activity. pQ3 beta HSD was able to reduce 3-keto steroids to their corresponding 3 beta-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3 beta HSD genes were isolated and sequenced from another 6 species of the genus Digitalis. Gene structure and the deduced 3 beta HSD proteins share a high degree of similarity.