Molecular cloning and heterologous expression of a progesterone 5β-reductase (5β-POR) from Isoplexis canariensis

Isoplexis (Lindl.) Lindl. ex Benth., endemic to the Canary Islands and Madeira, is a plant genus closely related to Digitalis L.. Isoplexis canariensis (L.) Lindl. ex. G. Don contains 5α- and 5β-cardenolides, together with cardenolides containing a Δ⁴ -or Δ⁵ -double-bond and saponins [1, 2]. The biosynthesis of cardenolides in Digitalis is well established [1, 3], whereas the biosynthesis in Isoplexis needs further investigation. A full-length cDNA clone that encodes progesterone 5β-reductase (5β-POR) was isolated from Isoplexis canariensis leaves. The reading frame of the 5β-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph I/Sal I 5β–POR fragment was cloned into the pQE vector system and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant His-tagged gene product was purified under native conditions on a Ni-nitrilotriacetic acid (Ni-NTA) matrix. Its size was determined by SDS-Page to be about 45 kDa. The purified recombinant protein was enzymatically active, as proven in a standard enzyme assay, using progesterone and NADPH as a substrate and cosubstrate, respectively. Biochemical parameters were determined; the K _m – and V_max -values for the putative natural substrate progesterone were calculated to be 0.215 mM and 46.4 nkat/mg protein, respectively. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were determined [4]. The 5β-POR from I. canariensis shares considerable homology with other progesterone 5β-reductases including those of various Digitalis species and Arabidopsis thaliana.

References: 1. Luckner, M., Wichtl, M. (2000), Digitalis, WVG Stuttgart. 2. Schaller, F., Kreis, W. (2006), Planta Med. submitted. 3. Kreis, W. et al. (1998), Planta Med. 64: 491–499. 4. Herl, V. et al. (2006), Planta Med. submitted.