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

The mustard family (Brassicaceae) supplies the world with many crop species and medicinal plants. In the Brassicaceae only few genera contain cardenolides (Syrenia, Draba, Erysimum incl. Cheiranthus) whereas all of them contain glucosinolates. One may ask whether cardenolides originated independently several times in the 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 cDNA encoding progesterone 5β-reductases (P5βR) from several Brassicaceae species (Armoracia rusticana, Aethionema grandiflora, Barbarea vulgaris, various Brassica species, Cakile maritima, Cardamine pratensis, Draba aizoides, Lunaria annua, Lepidium sativum, Matthiola tricuspidata, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale). P5βRs, are members of the short chain dehydrogenases/reductases (SDR) superfamily of proteins and are supposed to be involved in the biosynthesis of 5β-cardenolides. These enzymes have been characterized as substrate-promiscuous enone 1,4-reductases and are encoded by Vein Patterning 1 (VEP1) genes, which occur in a wide range of plant species independent of their ability to produce cardenolides or not. The nucleotide and deduced amino acid sequences of the new P5βRs were aligned with known and putative VEP1-encoded P5βRs and a cladistic tree was constructed which fitted well to the accepted Brassicaceae phylogenetic relationship, indicating a common ancestor for all Brassicaceae P5βRs. The new P5βR cDNAs were over-expressed in E. coli and the respective P5βR proteins tested for their catalytic function. Their kinetic constants were determined using progesterone and small enones as substrates. Molecular modelling was applied to elucidate structural and functional relationship between VEP1-encoded P5βRs.