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

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.

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
• 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
• 3 Trujillo J M, Hernandez O, Navarro E. Monoglycosides from Isoplexis chalchantha .  J Nat Prod. 1990;  53 167-70
• 4 Luckner M, Wichtl M. Digitalis . Stuttgart; WVGmbH 2000
• 5 Schaller F, Kreis W. Clonal propagation of Isoplexis canariensis (L.) Lindl.ex. G.Don.  Planta Med. 1996;  62 450-2
• 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
• 7 Kreis W, Hensel A, Stuhlemmer U. Cardenolide biosynthesis in foxglove.  Planta Med. 1998;  64 491-9
• 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
• 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
• 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
• 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
• 13 Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning. A Laboratory Manual. Vol. 3 New York; Cold Spring Harbor Laboratory Press 2001
• 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