VEP1- encoded enone 1,4-reductases from Brassicaceae: cloning, expression, molecular phylogeny and modelling

Progesterone 5β-reductases (P5βR), which are members of the short chain dehydrogenases/reductases (SDR) superfamily are involved in the biosynthesis of 5β-cardenolides. These enzymes have been characterized as substrate-promiscuous enone 1,4-reductases and are encoded by Vein Patterning1-like (VEP1) genes, which occur in cardenolide producing plants as well as in non-cardenolide producing plants. Since cardenolides have been reported for a few Brassicaceae genera only, one may ask whether cardenolides originated several times in Brassicaceae or whether they were lost during evolution in most of the genera. In order to shed light on the conservation and/or the multiple evolution of genes coding for cardenolide-biosynthetic enzymes, we isolated P5βRs from several Brassicaceae. The nucleotide and deduced amino acid sequences of the new P5βRs were aligned with known and putative VEP1-encoded P5βRs. The new genes were over-expressed in E. coli and the respective P5βR proteins tested for their catalytic function. Molecular modelling was applied to elucidate structural and functional relationship between VEP1-encoded P5βRs.