Progesterone 5ß-reductase of Erysimum crepidifolium: cDNA cloning, expression in Escherichia coli, and reduction of enones with the recombinant protein.

Erysimum is a genus of the Brassicaceae family closely related to the genus Arabidopsis. Several Erysimum species accumulate 5ß-cardenolides. Progesterone 5ß-reductases (P5ßRs) first described in Digitalis species are thought to be involved in 5ß-cardenolide biosynthesis. P5ßRs belong to the dehydrogenase/reductase super-family of proteins. A full length cDNA clone encoding a P5ßR was isolated from Erysimum crepidifolium leaves by 5'/3' RACE-PCR (termed EcP5ßR). Subsequently, the P5ßR cDNAs of another nine Erysimum species were amplified by RT-PCR using 5' and 3' end primers deduced from the EcP5ßR cDNA. The EcP5ßR cDNA is 1170bp long and encodes for 389 amino acids. The EcP5ßR cDNA was ligated into the vector pQE 30 UA and the recombinant His-tagged protein (termed rEcP5ßR) was over-expressed in Escherichia coli and purified by Ni-chelate affinity chromatography. Kinetic constants were determined for progesterone, 2-cyclohexen-1-one, isophorone, and NADPH. The by far highest specificity constant (k(cat)K(M)⁻¹) was estimated for 2-cyclohexen-1-one indicating that this monocyclic enone may be more related to the natural substrate of the enzyme than progesterone. The atomic structure of rEcP5ßR was modelled using the crystal structure of P5ßR from Digitalis lanata 2V6G as the template. All sequence motifs specific for SDRs as well as the NFYYxxED motif typical for P5ßR-like enzymes were present and the protein sequence fitted into the template smoothly.