Molecular Cloning and Expression of Progesterone 5β-Reductase (5β-POR) from Isoplexis canariensis

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A full-length cDNA clone that encodes progesterone 5β-reductase (5β-POR, EC 1.3.1.3) was isolated from Isoplexis canariensis leaves. The reading frame of the Ic5β-POR gene is 1170 nucleotides corresponding to 389 amino acids. The SphI/SalI Ic5β-POR fragment was cloned into the pQE vector system and then transformed into Escherichia coli strain M15[pREP4]. The gene was functionally expressed and the recombinant enzyme was characterised. K _m and V _max were calculated to be 0.215 mM and 46.4 nkat/mg protein, respectively, using progesterone as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The 5β-POR from I. canariensis shows a significant homology to the putative progesterone 5β-reductases isolated from other plant species, such as Digitalis lanata and Arabidopsis thaliana.

Isoplexis (Lindl.) Lindl. ex Benth. is a plant genus closely related to Digitalis L.. The members of the genus Isoplexis are regarded as relicts of the tertiary Macaronesian flora and are endemic to the Canary Islands and Madeira. Isoplexis canariensis (L.) Lindl. ex. G. Don contains cardenolides and saponins [1], [2], [3]. 5α-Cardenolides and cardenolides containing a Δ⁴- or Δ⁵-double bond have been described in three species whereas I. sceptrum (L.) Lindl. ex G. Don seems to be cardenolide-free (see [4] for more details). Recently, 5β-cardenolides of the digitoxin type have been reported to occur in all of the three cardenolide-containing members of the genus [5], [6], [7].

Progesterone 5β-reductase (5β-POR; Δ^{4 -} 3-oxosteroid 5β-reductase; EC 1.3.1.3) plays a crucial role in the biosynthesis of 5β-cardenolides in Digitalis lanata Ehrh. (see [8] for a review) since it catalyses the conversion of progesterone to 5β-pregnane-3,20-dione (Fig. [1]). The enzyme was partially sequenced [9] and cloned from D. purpurea [10] and the corresponding D. lanata gene was functionally expressed in E. coli [11]. The biosynthesis of cardenolides in Digitalis is well established [4], [8], while the biosynthesis in Isoplexis needs further investigation [11]. Here we report the cloning, functional expression and biochemical characterisation of 5β-POR from I. canariensis. Molecular and biochemical data are compared with those obtained for the recombinant D. lanata enzyme [11].

Leaves of I. canariensis were used for cDNA preparation. Primers were designed according to the known 5β-POR sequences. Gel electrophoresis showed a single DNA band of about 1250 bp. After sequencing the gene, the protein was identified and aligned with known and suggested 5β-POR genes from Digitalis lanata and Arabidopsis thaliana. The amino acid sequence of Ic5β-POR was approximately 98 % identical to D. lanata 5β-POR and about 69 % identical to the putative 5β-POR (AAL32529.1), derived from Arabidopsis thaliana (see Supporting Information).

Ic5β-POR has been successfully cloned into the expression system (pQE) for over-expression as His-Tag-fusion protein in Escherichia coli. Over-expression of recombinant Ic5β-POR was achieved through IPTG addition at low temperature. Following Ni-NTA batch fractionation, the purified enzyme was analysed by SDS-PAGE (Fig. [2]). The molecular mass of the recombinant 5β-POR was 45 kDa. Plant 5β-PORs discovered in D. purpurea and D. lanata had similar molecular masses [9], [11]. Minor compounds visible on SDS-PAGE (Fig. [2], Lane 6) were produced by E. coli [12].

The recombinant Ic5β-POR exhibited NADPH-dependent 5β-progesterone reductase activity. 5β-POR activity could not be detected in protein extracts from bacteria containing the pQE vector only. The kinetic properties of the r5β-POR were investigated. Relative activities as well as K _m value were calculated and are displayed in Table [1]. The K _m values for progesterone and NADPH were 215 μM and 15 μM, respectively. Other substrates, such as cortisol, cortexone and 4-androstene-3,17-dione, were also accepted by the recombinant enzyme. The carbonyl group at C-3 and the adjacent Δ⁴-double bond are essential structural requirements of Ic5β-POR substrates. The same conclusion was drawn in a previous study concerning the Digitalis 5β-POR [11].

Using progesterone as the substrate, pH and temperature dependences of the recombinant Ic5β-POR were investigated. The pH optimum was 8.5 and the enzyme worked best at around 45 °C. Its pI was 6.5 as determined by isoelectric focusing.

In summary, the 5β-POR isolated from I. canariensis differs only slightly from the corresponding Digitalis enzymes [9], [11]. Since 5α-cardenolides and cardenolides containing a Δ⁴- or Δ⁵-double bond occur side by side with 5β-cardenolides in Isoplexis, further studies are necessary to determine whether the biosynthesis of cardenolides follows identical pathways in Digitalis and Isoplexis and to identify the branching point leading to the various types (Δ⁴, Δ⁵, 5α, 5β) of cardenolides.

Materials and Methods

Plants of Isoplexis canariensis were grown under standard conditions in the Institute’s greenhouse [8]. A voucher specimen is deposited in the herbarium of the Institute of Biology at the Friedrich-Alexander-Universität, Erlangen-Nürnberg. Steroids were purchased from Steraloids INC (Newport, RI, USA). All other chemicals were from Sigma (Munich, Germany) or Merck (Darmstadt, Germany).

Plant tissue was ground to a fine powder in liquid nitrogen using a mortar and pestle. Genomic DNA and total RNA extraction was carried out with the E.Z.N.A.® Plant DNA and RNA Mini Kits (Peqlab GmbH; Erlangen, Germany). Messenger RNA was isolated using an Oligotex^TM direct mRNA Kit (QIAGEN GmbH; Hilden, Germany). All other molecular biology methods were performed according to [13].

PCR amplifications using SphI-primer (dir) 5′-ATATGCATGCAAAAAATGAGCTGGTGGTGG-3′ and SalI-primer (rev) 5′-TATAGTCGACAACCATGTCAAGGAACAATC-3′ were performed in a Personal Cycler T3 (Biometra GmbH; Göttingen, Germany). The PCR products were analysed as described earlier [11].

After the nucleotide sequence was determined (MWG Biotech AG; Martinsried, Germany), all data were analysed by different software packages (European Bioinformatics Institute). Sequence homology was analysed with the BLAST search tool using the GenBank data set. The nucleic acid sequences as well as the translated (TRANSEQ) amino acid sequences were aligned using ClustalW software. Recombinant Ic5β-POR was isolated in native form according to the manufacturer’s manual (QIAexpressionist; Qiagen; Hilden, Germany). The Ni-NTA matrix was washed with 20 mM imidazole buffer before elution by 250 mM imidazole buffer. Proteins were analysed in SDS-PAGE as reported earlier [11]. The activity of the Ic5β-POR was determined by TLC and HPLC [11], [14].

The isoelectric point (pI) for the r5β-POR was determined by isoelectric focusing on Criterion Gels IEF 3 - 9/Criterion Cell (BioRad; München, Germany). The temperature optimum was measured at pH 8.5. Kinetic constants were determined in the presence of 100 μg/mL enzyme and 0.3 μg substrate. Incubation time was 30 min.

The nucleotide sequences reported in this paper have been submitted to GenBank™ Data Base with the corresponding accession numbers: DQ218315 - 218 318.

Supporting information: Alignments of the deduced 5β-POR proteins from Isoplexis canariensis (DQ218315), Digitalis lanata (AY585867), and Arabidopsis thaliana (AAL32529.1) are available under ”Forschung” at http://www.biologie.uni-erlangen.de/pharmbiol/index.html

Acknowledgements

Plants, seeds and herbal material were kindly provided by Dr. W. Welss, Botanical Garden FAU Erlangen. Linguistic advice of Barbara White is gratefully acknowledged.

References

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  CrossrefPubMedSearch in Google Scholar
• 12 Janknecht R, de Martynoff G, Lou J, Hipskind R A, Noerdheim A, Stunnenberg H G. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant Vaccinia virus.  Proc Natl Acad Sci USA. 1991;  88 8972-6
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• 14 Egerer-Sieber C, Herl V, Müller-Uri F, Kreis W, Muller Y A. Crystallization and preliminary crystallographic analysis of selenomethionine-labeled progesterone 5beta-reductase from Digitalis lanata Ehrh.  Acta Crystallogr. 2006;  F62 186-8
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F. Müller-Uri

Lehrstuhl für Pharmazeutische Biologie

Friedrich-Alexander-Universität Erlangen-Nürnberg

Staudtstr. 5

91058 Erlangen

Germany

Phone: +49-9131-852-8251

Email: fmueller@biologie.uni-erlangen.de

References

• 1 Freitag H, Spengel S, Linde H H, Meyer K. The glycosides of the leaves from Isoplexis isabelliana (Webb) Masf.  Helv Chim Acta. 1967;  50 1336-6
  CrossrefPubMedSearch in Google Scholar
• 2 Gonzalez A G, Breton J L, Navarro E, Trujillo J, Boada J, Rodriguez R. Phytochemical study of Isoplexis chalchantha .  Planta Med. 1985;  51 9-11
  Thieme ConnectPubMedSearch in Google Scholar
• 3 Trujillo J M, Hernandez O, Navarro E. Monoglycosides from Isoplexis chalchantha .  J Nat Prod. 1990;  53 167-70
  CrossrefPubMedSearch in Google Scholar
• 4 Luckner M, Wichtl M. Digitalis . Stuttgart; WVGmbH 2000
  Search in Google Scholar
• 5 Schaller F, Kreis W. Clonal propagation of Isoplexis canariensis (L.) Lindl.ex. G.Don.  Planta Med. 1996;  62 450-2
  PubMedSearch in Google Scholar
• 6 Gavidia I, Seitz H U, Perez-Bermudez P, Vogler B. LC-NMR applied to the characterisation of cardiac glycosides from three micropropagated Isoplexis species.  Phytochem Anal. 2002;  13 266-71
  CrossrefPubMedSearch in Google Scholar
• 7 Kreis W, Hensel A, Stuhlemmer U. Cardenolide biosynthesis in foxglove.  Planta Med. 1998;  64 491-9
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Schaller F, Kreis W. Cardenolide genin pattern in Isoplexis plants and shoot cultures. Planta Med 2006, in press
• 9 Gärtner D E, Keilholz W, Seitz H U. Purification, characterization and partial peptide microsequencing of progesterone 5β-reductase from shoot cultures of Digitalis purpurea .  Eur J Biochem. 1994;  225 1125-32
  CrossrefPubMedSearch in Google Scholar
• 10 Roca-Perez L, Boluda R, Gavidia I, Perez-Bermudez P. Seasonal cardenolide production and Dop5 gene expression in natural populations of Digitalis obscura .  Phytochemistry. 2004;  65 869-78
  PubMedSearch in Google Scholar
• 11 Herl V, Fischer G, Müller-Uri F, Kreis W. Molecular cloning and heterologous expression of progesterone 5β-reductase from Digitalis lanata Ehrh.  Phytochemistry. 2006;  67 225-31
  CrossrefPubMedSearch in Google Scholar
• 12 Janknecht R, de Martynoff G, Lou J, Hipskind R A, Noerdheim A, Stunnenberg H G. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant Vaccinia virus.  Proc Natl Acad Sci USA. 1991;  88 8972-6
  PubMedSearch in Google Scholar
• 13 Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning. A Laboratory Manual. Vol. 3 New York; Cold Spring Harbor Laboratory Press 2001
  Search in Google Scholar
• 14 Egerer-Sieber C, Herl V, Müller-Uri F, Kreis W, Muller Y A. Crystallization and preliminary crystallographic analysis of selenomethionine-labeled progesterone 5beta-reductase from Digitalis lanata Ehrh.  Acta Crystallogr. 2006;  F62 186-8
  PubMedSearch in Google Scholar

F. Müller-Uri

Lehrstuhl für Pharmazeutische Biologie

Friedrich-Alexander-Universität Erlangen-Nürnberg

Staudtstr. 5

91058 Erlangen

Germany

Phone: +49-9131-852-8251

Email: fmueller@biologie.uni-erlangen.de