期刊
PHYTOCHEMISTRY
卷 71, 期 13, 页码 1495-1505出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.phytochem.2010.06.004
关键词
Digitalis; Plantaginaceae; Progesterone 5 beta-reductase; Enone reductase; Substrate promiscuity; Cardenolide
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. (C) 2010 Elsevier Ltd. All rights reserved.
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