Molecular cloning and heterologous expression of progesterone 5β-reductase from Digitalis lanata Ehrh.

A full-length cDNA clone that encodes progesterone 5 beta-reductase (5 beta-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5 beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5 beta-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The K,, and v,,,, values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg(-1) protein, respectively. Only 5 beta-pregnane-3,20-dione but not its alpha-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5 beta-POR gene in D. lanata was determined by Southern blot analysis. The 5 beta-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species. (c) 2005 Elsevier Ltd. All rights reserved.