Expression of 3β‐HSD and P5βR, Genes Respectively Coding for Δ5-3β-Hydroxysteroid Dehydrogenase and Progesterone 5β‐Reductase, in Leaves and Cell Cultures of Digitalis lanata EHRH

Mona Ernst; Rodrigo Maia de Padua; Vanessa Herl; Frieder Müller-Uri; Wolfgang Kreis

doi:10.1055/s-0030-1250007

Planta Medica, Inhaltsverzeichnis

Planta Med 2010; 76(9): 923-927
DOI: 10.1055/s-0030-1250007

Biochemistry, Molecular Biology and Biotechnology

Original Papers
© Georg Thieme Verlag KG Stuttgart · New York

Expression of 3β‐HSD and P5βR, Genes Respectively Coding for Δ⁵-3β-Hydroxysteroid Dehydrogenase and Progesterone 5β‐Reductase, in Leaves and Cell Cultures of Digitalis lanata EHRH[*]

Mona Ernst¹ , Rodrigo Maia de Padua¹ , Vanessa Herl² , Frieder Müller-Uri¹ , Wolfgang Kreis¹

¹Lehrstuhl für Pharmazeutische Biologie, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg,, Erlangen, Germany
²Tierärztliches Institut Göttingen, Göttingen, Germany

Abstract

Plants of the genus Digitalis produce 5β-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3β-Hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5β-reductase (P5βR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC‐MS methods. Expression of the respective genes, namely AY585867.1 (P5βR gene) and DQ466890.1 (3β‐HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5βR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.

Key words

Digitalis lanata - Plantaginaceae - cardiac glycosides - progesterone 5β‐reductase - Δ ⁵‐3β‐hydroxysteroiddehydrogenase - gene expression

Volltext

Referenzen

References

1 Kreis W, Müller-Uri F. Biochemistry of sterols, cardiac glycosides, brassinosteroids, phytoecdysteroids and steroid saponins. Wink M Annual plant reviews, Vol. 40. Biochemistry of plant secondary metabolism, 2nd edition. Sheffield; CRC Press 2010: 304-363
2 Hoppe U C, Böhm M, Dietz R, Hanrath P, Kroemer H K, Osterspey K, Schmaltz A A, Erdmann E. Leitlinien zur Therapie der chronischen Herzinsuffizienz. Z Kardiol. 2005; 94 488-509
3 Mathews C K, van Holde K E, Ahern K G. Biochemistry, 3rd edition. SanFrancisco; Addison-Wesley Publishing 2000: 340-342
4 Kreis W, Hensel A, Stuhlemmer U. Cardenolide biosynthesis in foxglove. Planta Med. 1998; 64 491-499
5 Gärtner D E, Wendroth S, Seitz H U. A stereospecific enzyme of the putative biosynthetic pathway of cardenolides. Characterization of a progesterone 5β-reductase from leaves of Digitalis purpurea L. FEBS Lett. 1990; 271 239-242
6 Seidel S, Kreis W, Reinhard E. 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. Plant Cell Rep. 1990; 8 621-624
7 Stuhlemmer U, Kreis W. Cardenolide formation and activity of pregnane-modifying enzymes in cell suspension cultures, shoot cultures and leaves of Digitalis lanata. Plant Physiol Biochem. 1996; 34 85-91
8 Lindemann P, Luckner M. Biosynthesis of pregnane derivatives in somatic embryos of Digitalis lanata. Phytochemistry. 1997; 46 507-513
9 Kreis W, Reinhard E. The production of secondary metabolites by plant cells cultivated in bioreactors. Planta Med. 1989; 55 409-416
10 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15 473-479
11 Blom T J M, Kreis W, van Iren F, Libbenga K R. A non-invasive method for the routine-estimation of fresh weight of cells grown in batch suspension cultures. Plant Cell Rep. 1992; 11 146-149
12 Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72 248-254
13 Herl V, Fischer G, Müller-Uri F, Kreis W. Molecular cloning and heterologous expression of progesterone 5β-reductase (P5βR) from Digitalis lanata EHRH. Phytochemistry. 2006; 67 225-231
14 Jork H. Thin layer chromatography: reagents and detection methods, Vol. 1. New York; VCH Publishers 1990: 195-198
15 Jensen K B. Paper chromatography of cardiac glycosides and aglycones from Digitalis purpurea. Acta Pharmacol Toxicol. 1953; 9 99-108
16 Kedde D L. Bijdrage tot het chemisch anderzoek van Digitalispreparaten. Pharm Weekbl. 1947; 82 741-757
17 Cordell G A, Lemos T L G, Monte F J Q, de Amttos M C. Vegetables as chemical reagents. J Nat Prod. 2007; 70 478-492
18 Herl V, Frankenstein J, Meitinger N, Müller-Uri F, Kreis W. Cloning, heterologous expression and characterization of Δ5-3β-hydroxsteroid dehydrogenase/Δ5-Δ4-ketosteroid isomerase from Digitalis lanata EHRH. Planta Med. 2007; 73 704-710
19 Schebitz P, Nothdurft L, Hensel A, Müller-Uri F, Kreis W. Norcholanic acids as substrates for recombinant 3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase, enzymes of the 5β-cardenolide biosynthesis. Tetrahedron Lett. 2010; 51 367-370
20 Finsterbusch A, Lindemann P, Grimm R, Eckerskorn C, Luckner M. Δ5-3β-Hydroxsteroid dehydrogenase from Digitalis lanata EHRH. – a multifunctional enzyme in steroid metabolism?. Planta. 1999; 209 478-486
21 Gärtner D E, Seitz H U. Enzyme activities in cardenolide-accumulating, mixotrophic shoot cultures of Digitalis purpurea L. J Plant Physiol. 1993; 141 269-275
22 Herl V, Fischer G, Bötsch R, Müller-Uri F, Kreis W. Molecular cloning and expression of progesterone 5β-reductase (P5βR) from Isoplexis canariensis. Planta Med. 2006; 72 1163-1165
23 Herl V, Fischer G, Reva V A, Stiebritz M, Muller Y A, Müller-Uri F, Kreis W. The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-Δ4,5-steroid 5β-reductase activity in Arabidopsis thaliana L. Biochimie. 2009; 91 517-525
24 Witt M. Klonierung, Expression, Reinigung und Charakterisierung von 3β-Hydroxsteroid-Dehydrogenasen aus Arabidopsis thaliana (L.) HEYNHOLD. [dissertation]. Wittenberg; Universität Halle-Wittenberg 2008
25 Perez-Bermudez P, Moya Garcia A A, Tunon I, Gavidia I. Digitalis purpurea P5βR2, encoding steroid 5β-reductase, is a novel defense-related gene involved in cardenolide biosynthesis. New Phytol. 2010; 185 687-700
26 Herl V, Albach D C, Müller-Uri F, Bräuchler C, Heubl G, Kreis W. Using progesterone 5β-reductase, a gene encoding a key enzyme in the cardenolide biosynthesis, to infer the phylogeny of the genus Digitalis. Plant Syst Evol. 2008; 271 65-78
27 Roca-Perez L, Boluda R, Gavidia I, Perez-Bermudez P. Seasonal cardenolide production and Dop5βr gene expression in natural populations of Digitalis obscura. Phytochemistry. 2004; 65 1869-1878
28 Jun J H, Ha C M, Nam H G. Involvement of the VEP1 gene in vascular strand development in Arabidopsis thaliana. Plant Cell Physiol. 2002; 43 323-330
29 Thorn A, Egerer-Sieber C, Jäger C M, Herl V, Müller-Uri F, Kreis W, Muller Y. The crystal structure of progesterone 5β-reductase from Digitalis lanata defines a novel class of short-chain dehydrogenases/reductases. J Biol Chem. 2008; 283 17260-17269
30 Burda E, Krausser M, Fischer G, Hummel W, Müller-Uri F, Kreis W, Gröger H. Recombinant Δ4,5-steroid 5β-reductases as biocatalysts for the reduction of activated C=C-double bonds in monocyclic and acyclic molecules. Adv Synth Catal. 2009; 351 2787-2790

1 Dedicated to Prof. Dr. Wolfgang Barz on the occasion of his 75^th birthday.

Prof. Dr. Wolfgang Kreis

Lehrstuhl für Pharmazeutische Biologie
Department Biologie
Friedrich-Alexander-Universität Erlangen-Nürnberg

Staudtstrasse 5

91058 Erlangen

Germany

Telefon: + 49 9 13 18 52 82 41

Fax: + 49 9 13 18 52 82 43

eMail: wkreis@biologie.uni-erlangen.de

Expression of 3β‐HSD and P5βR, Genes Respectively Coding for Δ5-3β-Hydroxysteroid Dehydrogenase and Progesterone 5β‐Reductase, in Leaves and Cell Cultures of Digitalis lanata EHRH[*]

Expression of 3β‐HSD and P5βR, Genes Respectively Coding for Δ⁵-3β-Hydroxysteroid Dehydrogenase and Progesterone 5β‐Reductase, in Leaves and Cell Cultures of Digitalis lanata EHRH[*]