Δ5-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism?

Δ⁵-3β-Ηydroxysteroid dehydrogenase (Δ⁵-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ⁵-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ⁵-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed. Received: 20 January 1999 / Accepted: 5 May 1999     

Δ5-3β-hydroxysteroid dehydrogenase/Δ5-Δ4-ketosteroid isomerase (3β-HSD), a possible enzyme of cardiac glycoside biosynthesis,in cell cultures and plants of Digitalis lanata EHRH

Summary The in vitro transformation of pregnenolone into progesterone in Digitalis lanata tissues was shown to be catalyzed by a 3-hydroxysteroid dehydrogenase/ketosteroid isomerase (3-HSD). Product formation was monitored by HPLC. The enzyme could be partially characterized and 3-HSD activities were measured in various Digitalis lanata tissues and in cell cultures of other plant species. Since no correlation was observed between biosynthetic competence of the tissue and 3-HSD activity, it was concluded that this enzyme does not play a major role in regulating cardenolide biosynthesis.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - DMF N,N-dimethylformamide - IAA indole-3-acetic acid     

The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-Δ-steroid 5β-reductase activity in Arabidopsis thaliana L.

The Arabidopsis thaliana VEP1 gene product shows about 70% sequence identity to Digitalis lanata progesterone 5β-reductase, an enzyme considered to catalyze a key step in the biosynthesis of cardiac glycosides. A. thaliana does not accumulate cardenolides but protein extracts prepared from its leaves were capable of reducing progesterone to 5β-pregnane-3,20-dione. A full-length cDNA clone encoding a Δ^4,5-steroid 5β-reductase (At5β-StR, EC 1.1.1.145/1.3.1.23), a member of the short-chain dehydrogenase/reductase (SDR) family, was isolated from A. thaliana leaves. A SphI/SalI At5β-StR gene fragment was cloned into the pQE vector system and transformed into Escherichia coli. The gene was functionally expressed and the recombinant His-tagged fusion protein was characterized. K_m values and specific activities for putative 3-oxo-Δ^4,5-steroid substrates such as progesterone, cortisol, cortexone and 4-androstene-3,17-dione, and for the co-substrate NADPH were determined. Progesterone was stereo-specifically reduced to 5β-pregnane-3,20-dione and none of the 3-oxo-Δ^5,6-steroids tested were accepted as a substrate. The gene encoding At5β-StR was strongly transcribed in stems and leaves. A three-dimensional model of At5β-StR highlights a close structural similarity to the related, previously described D. lanata progesterone 5β-reductase. This homology extends to the active site where single amino acid substitutions might be responsible for the increased catalytic efficiency of At5β-StR when compared to the activity of the recombinant form of the D. lanata enzyme.     

Identification of a putative DNA replication origin in the λ-aminobutyric acid receptor subunit β3 and α5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing

The region containing the GABA_A receptor β3 and α5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone λ84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage λ84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13 kb of contiguous sequence reveals several A +T-rich regions which display potential DNA unwinding. The plasmid subclones from phage λ84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABA_A receptor subunit gene cluster.     

Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: Mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3β-hydroxy-steroid dehydrogenase/Δ–Δ isomerase pathway

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3β-hydroxy-steroid dehydrogenase/Δ⁵–Δ⁴ isomerase (3β-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3β-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3β-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13α-methyl analogue. In contrast to previous studies of fungi with 3β-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one were metabolized solely through the putative 3β-HSD/isomerase pathway, indicating that a 17β-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3β-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.