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

Abstract

During the biosynthesis of cardiac glycosides, Δ⁵-3β-hydroxysteroid dehydrogenase (3βHSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3β-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3βHSD 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 3βHSD was isolated by RT-PCR from the same source. A Sph I/Kpn I 3βHSD cDNA was cloned into the pQE30 vector and then transferred into E. coli strain M15[pREP4]. 3βHSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3βHSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). pQ3βHSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3β-hydroxypregnanes but not cholesterol were 3β-oxidised by pQ3βHSD when NAD was used as the co-substrate. Testosterone (4-androsten-17β-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3βHSD has also 17β-dehydrogenase activity. pQ3βHSD was able to reduce 3-keto steroids to their corresponding 3β-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3βHSD genes were isolated and sequenced from another 6 species of the genus Digitalis. Gene structure and the deduced 3βHSD proteins share a high degree of similarity.

Key words

Δ⁵-3β-Hydroxysteroid dehydrogenase - Δ⁵-3-ketosteroid isomerase - Digitalis lanata - cardenolide biosynthesis - gene expression - Plantaginaceae - pregnenolone - progesterone

1 The nucleotide sequences reported in this paper have been submitted to GenBank™ Data Base with the corresponding accession numbers: DQ466890; AY844960; AY789449-453; AY844959.

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1 The nucleotide sequences reported in this paper have been submitted to GenBank™ Data Base with the corresponding accession numbers: DQ466890; AY844960; AY789449-453; AY844959.