Progesterone 5␤-reductase (5␤-POR) catalyzes the stereospecific reduction of progesterone to 5␤-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5␤-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5␤-POR from Digitalis lanata in complex with NADP ؉ at 2.3 Å and without cofactor bound at 2.4 Å resolution together with a model of a ternary complex consisting of 5␤-POR, NADP ؉ , and progesterone. Indeed, 5␤-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5␤-POR reduces a conjugated double bond in a steroid substrate via a 1-4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5␤-POR defines a novel class of SDRs.The beneficial effects of cardenolides, also known as cardiac glycosides or cardiotonic steroids, are well documented, and they have been applied for the treatment of cardiac insufficiencies for centuries (1-3). On a molecular level, these steroids are potent inhibitors of the sodium/potassium pump (Na ϩ /K ϩ -ATPase) that is present in almost all cells in higher organisms (4). Digitalis plants are still the major source for cardenolides, and as a step in the biosynthetic pathway, the Digitalis enzyme progesterone 5␤-reductase (5␤-POR) 2 catalyzes the stereospecific NADPH-dependent reduction of the ⌬ 4 -double bond in progesterone to 5␤-pregnane-3,20-dione. Because all Digitalis cardenolides share the characteristic 5␤-configuration, the enzyme 5␤-POR catalyzes a central step during their biosynthesis (5-7).NADH/NADPH-dependent reductases as well as the related dehydrogenases, dehydratases, and epimerases can be classified into two major protein families: the (␣/␤) 8 -barrel containing aldo-keto-reductases (AKRs) (8) and the Rossman fold containing short chain dehydrogenases/reductases (SDRs) (9 -11). Additional families such as the long and medium chain dehydrogenases/reductases are related to SDRs because they share with the latter the dinucleotide-binding double Rossman fold (12)(13)(14). SDRs are about 250 residues long and form a large family with over 2000 members (15). Because their central feature consists of an all-p...

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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.

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