Molecular cloning and expression of progesterone 5beta-reductase (5beta-POR) from Isoplexis canariensis

A full-length cDNA clone that encodes progesterone 5beta-reductase (5beta-POR, EC 1.3.1.3) was isolated from ISOPLEXIS CANARIENSIS leaves. The reading frame of the IC5beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. The SPHI /SALI IC5beta-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 5beta-POR from I. CANARIENSIS shows a significant homology to the putative progesterone 5beta-reductases isolated from other plant species, such as DIGITALIS LANATA and ARABIDOPSIS THALIANA.