title: Highly conserved progesterone 5 beta-reductase genes (P5 beta R) from 5 beta-cardenolide-free and 5 beta-cardenolide-producing angiosperms.
abstract: Most cardenolides used in the therapy of cardiac insufficiency are 5 beta-configured and thus the stereo-specific reduction of the Delta(4,5)-double bond of a steroid precursor is a crucial step in their biosynthesis. This step is thought to be catalysed by progesterone 5 beta-reductases. We report here on the isolation of 11 progesterone 5 beta-reductase (P5 beta R) orthologues from 5 beta-cardenolide-free and 5 beta-cardenolide-producing plant species belonging to five different angiosperm orders (Brassicales, Gentianales, Lamiales, Malvales and Solanales). Amino acid sequences of the P5 beta R described here were highly conserved. They all contain certain motifs qualifying them as members of a class of stereo-selective enone reductases capable of reducing activated C=C double bonds by a 1,4-addition mechanism. Protein modeling revealed seven conserved amino acids in the substrate-binding/catalytic site of these enzymes which are all supposed to exhibit low substrate specificity. Eight P5 beta R genes isolated were expressed in Escherichia coli. Recombinant enzymes reduced progesterone stereo-specifically to 5 beta-pregane-3,20-dione. The progesterone 5 beta-reductases from Digitalis canariensis and Arabidopsis thaliana reduced activated C=C double bonds of molecules much smaller than progesterone. The specific role of progesterone 5 beta-reductases of P5 beta Rs in cardenolide metabolism is challenged because this class of enone reductases is widespread in higher plants, and they accept a wide range of enone substrates.
authors.affiliation: Department of Biology, University of Erlangen-Nuremberg, Staudtstr. 5, 91058 Erlangen, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Bauer
authors.forename: Jennifer
authors.initials: J
authors.lastname: Munkert
authors.forename: Margareta
authors.initials: M
authors.lastname: Brydziun
authors.forename: Edyta
authors.initials: E
authors.lastname: Burda
authors.forename: Frieder
authors.initials: F
authors.lastname: Müller-Uri
authors.forename: Harald
authors.initials: H
authors.lastname: Gröger
authors.forename: Yves A
authors.initials: YA
authors.lastname: Muller
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Kreis
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D002298:Cardenolides / Q000378:metabolism*; D020134:Catalytic Domain; D017124:Conserved Sequence; D019684:Magnoliopsida / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny
chemical_list: D002298:Cardenolides; D010088:Oxidoreductases; C090493:progesterone 5 beta-reductase