Delta 5-3beta-hydroxysteroid dehydrogenase (3 beta HSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme.
During the biosynthesis of cardiac glycosides, Delta (5)-3beta-hydroxysteroid dehydrogenase (3 beta HSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3beta-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3 beta HSD 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 I /KPN I 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 3beta-hydroxypregnanes but not cholesterol were 3beta-oxidised by pQ3 beta HSD when NAD was used as the co-substrate. Testosterone (4-androsten-17beta-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3 beta HSD has also 17beta-dehydrogenase activity. pQ3 beta HSD was able to reduce 3-keto steroids to their corresponding 3beta-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.
Herl
Vanessa
V
Lehrstuhl für Pharmazeutische Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Frankenstein
Jördis
J
Meitinger
Nadine
N
Müller-Uri
Frieder
F
Kreis
Wolfgang
W
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D000595:Amino Acid Sequence; D018744:DNA, Plant / Q000032:analysis; D004070:Digitalis / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D008517:Phytotherapy; D018515:Plant Leaves; D016133:Polymerase Chain Reaction; D012639:Seeds; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018744:DNA, Plant; D015096:3-Hydroxysteroid Dehydrogenases; C031954:delta(5)-3 beta-hydroxysteroid dehydrogenase