Cloning, expression and structural investigations of P5βR-like enone reductases from various

5β-configured cardenolides are of great medicinal and economical importance in the treatment of cardiac insuffiency in humans. Considering their biosynthesis the stereospecific reduction of progesterone to 5β-pregane-3,20-dion is often referred to as key step, because here the characteristic cis configuration between ring A and B is formed. The reaction is catalysed by the enzyme progesterone 5β-reductase (P5βR), which has been cloned from several members of the genus Digitalis and functionally expressed in E. coli [1]. The crystal structure of Digitalis lanata P5βR has been solved revealing a novel class of short chain dehydrogenases/reductases (SDRs), with only two of the typical catalytic residues (K147 and Y179) being conserved [2]. An orthologous gene (VEP1) has also been cloned from Arabidopsis thaliana, which does not produce cardenolides. The respective recombinant enzyme was also capable of reducing progesterone stereoselectively [3]. Both enzymes were capable of reducing other steroidal, cyclic or noncyclic enone substrates [4].

Here we report the cloning of orthologous enzymes from various cardenolidecontaining and cardenolidefree medicinal important angiosperms and their functional expression in E. coli. We used the D. lanata 5β-POR structure (PDB 2v6g) as a template for modeling. We included the substrate progesterone into the 3D models and compared the bindingsites of the functional active enzymes. In addition to the expected catalytic residues another five amino acids could be identified within the substrate-binding pocket being conserved in all enzymes. The importance of the structurally conservation of these residues will now be investigated using sitedirected mutagenesis.

References: 1. Herl, V. et al. (2006) Phytochemistry 67: 225231.

2. Thorn, A. et al. (2008)J. Biol. Chem 283: 1726017269.

3. Herl, V. et al. (2009) Biochimie 91: 517525.

4. Burda, E. et al. (2009) Adv. Synth. Catal. 351: 27872790.