Wolfgang Kreis

The Crystal Structure of Progesterone 5β-Reductase from Digitalis lanata Defines a Novel Class of Short Chain Dehydrogenases/Reductases

Progesterone 5β-reductase (5β-POR) catalyzes the stereospecific reduction of progesterone to 5β-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5β-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 5β-POR from Digitalis lanata in complex with NADP+ at 2.3Å and without cofactor bound at 2.4Å resolution together with a model of a ternary complex consisting of 5β-POR, NADP+, and progesterone. Indeed, 5β-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 5β-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 5β-POR defines a novel class of SDRs.

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16′-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, α -acetyldigitoxin, and α-acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.

Selective Uptake and Vacuolar Storage of Primary Cardiac Glycosides by Suspension-cultured Digitalis lanata Cells

Summary Cardenolide uptake by Digitalis cells and protoplasts was examined. The uptake of secondarycardiac glycosides (digitoxin, digoxin, β-methyldigitoxin, β-methyldigoxin) was not affected by ATPase inhibitors, uncoupling agents, or electron transport inhibitors and was thus inferred to result from simple diffusion of the relatively hydrophobic glycosides across the plasma-lemma. The uptake of primary glycosides (purpurea-glycoside A, lanatoside A, deacetyl-lanatoside C, lanatoside C), on the other hand, was inhibited reversibly by uncoupling agents and electron transport inhibitors. It was not affected by ATPase inhibitors. Cell cultures from plant species which do not synthesize cardenolides were unable to accumulate primary cardiac glycosides. Wash-out studies with Digitalis cells and protoplasts showed that secondary glycosides were easily released into the culture medium, while the primary glycosides were retained by the cells. To investigate the intracellular distribution of cardiac glycosides, vacuoles were prepared from cardenolide-containing Digitalis cells and analysed for their cardenolide content using an HPLC method. The primary glycosides were located almost exclusively in the vacuoles. A model for cardenolide uptake, transformation, and transport across the tonoplast is proposed.

Cardenolide glucosyltransferases and glucohydrolases in leaves and cell cultures of three Digitalis (Scrophulariaceae) species

Summary The enzymatic synthesis and hydrolysis of primary cardiac glycosides in leaves and suspension cultures of Digitalis lanata , D. purpurea and D. heywoodii were investigated. Cardenolide glucosyltransferase, with an activity ranging from 0.1 to 21.5µkat/kg protein, was demonstrated in leaves, callus and suspension-cultured cells of all three species examined. Cardenolide-hydrolysing glucosidases (digilanidases) were solubilized from leaves of the Digitalis species using detergent-containing buffers. The specific enzyme activities in buffered leaf extracts reached 1 to 5 mkat/kg. The properties of the cardenolide glucohydrolases from Digitalis lanata , D. heywoodii , and D. purpurea were compared. Optimal enzyme activities occurred at around pH 4.5 and 50 – 60°C. Considerable variations in substrate preferences were observed among the cardenolide glucosidases of the three species. A method was devised to determine cardenolide glucosyltransferase in tissues with high digilanidase activity. Extraction with detergent-free buffers containing 10mM D-gluconic acid-l,5-lactone yielded crude enzyme extracts in which less than 1% digilanidase activity, compared to a control, could be detected. Under the same conditions the glucosyltransferase still reached activity levels of about 90 % of that in controls without gluconic acid-l,5-lactone.

Iridoid Synthase Activity Is Common among the Plant Progesterone 5β-Reductase Family

Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterization of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2, and CrP5βR4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. We cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5βR proteins and has evolved early during evolution.

Highly conserved progesterone 5β-reductase genes (P5βR) from 5β-cardenolide-free and 5β-cardenolide-producing angiosperms

Most cardenolides used in the therapy of cardiac insufficiency are 5 beta-configured and thus the stereo-specific reduction of the Delta(4,5)-double bond of a steroid precursor is a crucial step in their biosynthesis. This step is thought to be catalysed by progesterone 5 beta-reductases. We report here on the isolation of 11 progesterone 5 beta-reductase (P5 beta R) orthologues from 5 beta-cardenolide-free and 5 beta-cardenolide-producing plant species belonging to five different angiosperm orders (Brassicales, Gentianales, Lamiales, Malvales and Solanales). Amino acid sequences of the P5 beta R described here were highly conserved. They all contain certain motifs qualifying them as members of a class of stereo-selective enone reductases capable of reducing activated C=C double bonds by a 1,4-addition mechanism. Protein modeling revealed seven conserved amino acids in the substrate-binding/catalytic site of these enzymes which are all supposed to exhibit low substrate specificity. Eight P5 beta R genes isolated were expressed in Escherichia coli. Recombinant enzymes reduced progesterone stereo-specifically to 5 beta-pregane-3,20-dione. The progesterone 5 beta-reductases from Digitalis canariensis and Arabidopsis thaliana reduced activated C=C double bonds of molecules much smaller than progesterone. The specific role of progesterone 5 beta-reductases of P5 beta Rs in cardenolide metabolism is challenged because this class of enone reductases is widespread in higher plants, and they accept a wide range of enone substrates.

Antiherpes activity of glucoevatromonoside, a cardenolide isolated from a Brazilian cultivar of Digitalis lanata

Cardiac glycosides, known ligands of the sodium pump, are widely used in the treatment of heart failure, such as digoxin and digitoxin. Besides this important activity, other biological activities, such as the antiviral activity, have been described for this group. HSV are responsible for many infections of oral, ocular and genital regions. Treatment with nucleoside analogs such as acyclovir is effective in most cases; however drug-resistance may arise due to prolonged treatment mainly in immunocompromised individuals. In this study, an antiherpes screening was performed with 65 cardenolide derivatives obtained from different sources, and one natural cardenolide, glucoevatromonoside, inhibited HSV-1 and HSV-2 replication at very low concentrations. This cardenolide showed viral inhibitory effects if added up to 12h p.i. and these effects appear to take place by the inhibition of viral proteins synthesis (ICP27, U(L)42, gB, gD), the blockage of virus release and the reduction of viral cell-to-cell spread. This compound also showed synergistic antiviral effects with acyclovir and anti-Na(+)K(+)ATPase activity, suggesting that cellular electrochemical gradient alterations might be involved in the mechanism of viral inhibition. These results suggest that cardenolides might be promising for future antiviral drug design.

Catecholamine Biosynthesis Dependent on the Dark in Betacyanin-forming Portulaca Callus

Betacyanin-forming callus of the red-flowering (var. JR) Portulaca grandiflora, Hook, is shown to contain dopamine, adrenaline, noradrenaline, and epinine. Whereas the synthesis of DOPA, dopamine, and betacyanin is much higher in callus kept in the dark than in illuminated callus, the other catecholamines are only accumulated in the dark. The synthesis of dopamine via DOPA can be demonstrated by the use of inhibitors.

The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-Δ4,5-steroid 5β-reductase activity in Arabidopsis thaliana L.

The Arabidopsis thaliana VEP1 gene product shows about 70% sequence identity to Digitalis lanata progesterone 5beta-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 5beta-pregnane-3,20-dione. A full-length cDNA clone encoding a Delta(4,5)-steroid 5beta-reductase (At5beta-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 At5beta-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-Delta(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 5beta-pregnane-3,20-dione and none of the 3-oxo-Delta(5,6)-steroids tested were accepted as a substrate. The gene encoding At5beta-StR was strongly transcribed in stems and leaves. A three-dimensional model of At5beta-StR highlights a close structural similarity to the related, previously described D. lanata progesterone 5beta-reductase. This homology extends to the active site where single amino acid substitutions might be responsible for the increased catalytic efficiency of At5beta-StR when compared to the activity of the recombinant form of the D. lanata enzyme.

Separation of cardiac glycosides by micellar electrokinetic capillary electrophoresis

The separation of mixtures of primary and secondary cardiac glycosides by micellar electrokinetic capillary electrophoresis modified by cyclodextrins, urea and sodium cholate proved to be suitable for the determination of these hydrophobic compounds. It was possible to distinguish the two anomeric cardenolides glucodigifucoside and glucodigiglucomethyloside with all three buffer systems. Electropherograms of crude plant cell extracts from Digitalis lanata were obtained with this method.