Δ⁵-3β-Hydroxysteroid Dehydrogenase (3βHSD) from Digitalis lanata. Heterologous Expression and Characterisation of the Recombinant Enzyme[*]

Abstract

During the biosynthesis of cardiac glycosides, Δ⁵-3β-hydroxysteroid dehydrogenase (3βHSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3β-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3βHSD 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 3βHSD was isolated by RT-PCR from the same source. A Sph I/Kpn I 3βHSD cDNA was cloned into the pQE30 vector and then transferred into E. coli strain M15[pREP4]. 3βHSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3βHSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). pQ3βHSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3β-hydroxypregnanes but not cholesterol were 3β-oxidised by pQ3βHSD when NAD was used as the co-substrate. Testosterone (4-androsten-17β-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3βHSD has also 17β-dehydrogenase activity. pQ3βHSD was able to reduce 3-keto steroids to their corresponding 3β-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3βHSD genes were isolated and sequenced from another 6 species of the genus Digitalis. Gene structure and the deduced 3βHSD proteins share a high degree of similarity.

Δ⁵-3β-Hydroxysteroid dehydrogenase - Δ⁵-3-ketosteroid isomerase - Digitalis lanata - cardenolide biosynthesis - gene expression - Plantaginaceae - pregnenolone - progesterone

References

• 1 Luckner M, Wichtl M. In: Luckner M, Wichtl M, editors Digitalis. Stuttgart; WVGmbH 2000: 1-352.
• 2 Kreis W, Hensel A, Stuhlemmer U. Cardenolide biosynthesis in foxglove.  Planta Med. 1998;  64 491-9.
• 3 Seidel S, Kreis W, Reinhard E. Δ⁵-3β-Hydroysteroid 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;  8 621-4.
• 4 Finsterbusch A, Lindemann P, Grimm R, Eckerskorn C, Luckner M. Δ⁵-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh - a multifunctional enzyme in steroid metabolism?.  Planta. 1999;  209 479-86.
• 5 Lindemann P, Finsterbusch A, Pangert A, Luckner M. Partial cloning of a Δ^{5 -} 3β-hydroxysteroid dehydrogenase from Digitalis lanata . In: Okamoto M, Ishimura Y, Nawata, H, editors Molecular steroidogenesis, Proceedings of the Yamada Conference LII. Tokyo; Universal Academy Press, Frontiers Science Series 29 - XXIV 2000: 333-4.
• 6 Simard J, Ricketts M -L, Gingras S, Soucy P, Feltus F A, Melner M H. Molecular biology of the 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase gene family.  Endocrine Rev. 2005;  26 525-82.
• 7 Persson B, Krook M, Jörnvall H. Characteristics of short-chain alcohol dehydrogenases and related enzymes.  Eur J Biochem. 1991;  200 537-43.
• 8 Djerassi C, Engle R R, Bowers A. The direct conversion of steroidal Δ⁵-3β-alcohols to Δ⁵- and Δ⁴-3-ketones.  J Org Chem. 1956;  21 547-9.
• 9 Kreis W, Zhu W, Reinhard E. 12β-hydroxylation of digitoxin by Digitalis lanata cells. Production of deacetyllanatoside C in a 20-L airlift bioreactor.  Biotechnol Lett. 1989;  11 25-30.
• 10 Kreis W, Reinhard E. 12β-hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells. Production of deacetyllanatoside C using a two-stage culture method.  Planta Med. 1988;  54 95-100.
• 11 Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning. A laboratory manual, 2nd edition. New York; Cold Spring Harbor Press 1989.
• 12 Williams B, Tsang A. A maize gene expressed during embryogenesis is ABA-inducible and highly conserved.  Plant Mol Biol. 1991;  16 919-23.
• 13 Müller-Uri F, Reva V A. Overexpression and catalytic function of cyclophilin 18 from Digitalis lanata Ehrh.  Pharm Pharmacol Lett. 2000;  10 5-7.
• 14 Herl V, Fischer G, Müller-Uri F, Kreis W. Molecular cloning and heterologous expression of progesterone 5β-reductase from Digitalis lanata Ehrh.  Phytochemistry. 2006;  67 25-31.
• 15 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;  72 248-54.
• 16 Jork H. Thin Layer Chromatography: Reagents and Detection Methods, Vol. 1. In: Jork H., Funk W, Fischer W, editors. Thin-Layer-Chromatography.  New York: VCH. Publishers;  2004 195-8.
• 17 Ringer K L, Davis E M, Croteau R. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.  Plant Physiol. 2005;  137 863-72.
• 18 Xia Z -Q, Costa M A, Pélissier H C, Davin L B, Lewis N G. Secoisolariciresinol dehydrogenase purification, cloning, and functional expression.  J Biol Chem. 2001;  276 12 614-23.
• 19 Jörnvall H, Persson B, Krook M, Atrian S, Gonzalez-Duarte R, Jeffery J. et al . Short-chain dehydrogenases/reductases (SDR).  Biochemistry. 1995;  34 6003-13.
• 20 Jez J M, Bennett M J, Schlegel B P, Lewis M, Penning T M. Comparative anatomy of the aldo-keto reductase superfamily.  Biochem J. 1997;  326 625-36.
• 21 Oppermann U CT, Maser E. Characterization of a 3α-hydroxysteroid dehydrogenase/carbonyl reductase from the gram-negative bacterium Commamonas testosteroni .  Eur J Biochem. 1996;  209 459-66.
• 22 Oppermann U CT, Filling C, Berndt K D, Persson B, Benach J, Ladenstein R. et al . Active site directed mutagenesis of 3β/17β-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions.  Biochemistry. 1997;  36 34-40.
• 23 Thomas J L, Berko E A, Faustino A, Myers R P, Strickler R C. Human placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5-4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids.  J Steroid Biochem. 1988;  31 785-93.
• 24 Benach J, Filling C, Oppermann U C, Roversi P, Bricogne G, Berndt K D. et al . Structure of bacterial 3β/17β-hydroxysteroid dehydrogenase at 1.2 A resolution: a model for multiple steroid recognition.  Biochemistry. 2002;  41 4659-68.
• 25 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
• 26 Seitz H U, Gärtner D E. Enzymes is cardenolide-accumulating shoot cultures of Digitalis purpurea L.  Plant Cell Tissue Organ Cult. 1994;  38 337-44.
• 27 Pollack R M. Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase.  Bioorg Chem. 2004;  32 341-53.
• 28 Houck W J, Pollack R M. Activation enthalpies and entropies for the microscopic rate constants of acetate-catalyzed isomerization of 5-androstene-3,17-dione.  J Am Chem Soc. 2003;  125 10 206-12.
• 29 Krozowski Z. The short-chain alcohol dehydrogenase superfamily: variations on a common theme. J.  Steroid Biochem Mol Biol. 1994;  51 125-30.

1 The nucleotide sequences reported in this paper have been submitted to GenBank™ Data Base with the corresponding accession numbers: DQ466890; AY844960; AY789449-453; AY844959.