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BackgroundProgesterone 5β-reductases (P5ßRs) catalyse one step in the stereospecific biosynthesis of cardenolides (potent repellents of herbivores and pharmaceutical stimulants of disordered heart muscle cells). They were originally discovered in the genus Digitalis and have since been frequently isolated from other angiosperms. Recombinant P5ßRs engineered in Escherichia coli host cells convert a broad spectrum of compounds in vitro, sometimes with higher efficiency than with progesterone. This observation suggests additional functions for cardenolide metabolism and promises future use in sustainable chemistry and biotechnology.MethodsA tissue complementary DNA (cDNA) library was screened for orthologous P5ßRs. Candidates were subcloned into expression vectors and overexpressed in E. coli cells. The recombinant P5ßR protein was investigated for catalytic activity with several related substrates. Using spectrophotometric assays, the biochemical parameters of the enzyme were calculated. A 3D model was created and was compared to the previously published P5ßR structure of Digitalis lanata and other plant P5ßR models.ResultsPerforming protein similarity searches in public databases and comparison of 3D protein structure models revealed four cDNA clones in a tissue library of Picea sitchensis (Bong.) Carrière putatively encoding P5ßRs. Succeeding with the expression of one clone in E. coli, the highly purified protein was unambiguously able to enantioselectively convert progesterone into 5ß-pregnane-3,20-dione. However, the catalytic activity to reduce the small molecule 2-cyclohexen-1-one was nearly 100 times faster. Methyl vinyl ketone was reduced similar to results from previously studied angiosperm resources.Conclusions(i) The low catalytic efficiency for progesterone conversion agrees with the fact that conifers have not been reported to accumulate cardenolides. This finding suggests that alternate metabolic processes occur whereby the newly detected enzymes could transform smaller molecules rather than large ones such as progesterone. (ii) An ancient P5ßR gene appears to have existed in the last common ancestor of seed plants approximately 300 million years ago. If the diversification of P5ßRs, including the currently detected homologous iridoid synthase activity, was related to stress encountered during the transition to growth on land, then investigation of P5ßRs from pteridophytes and bryophytes should improve our knowledge of this enzyme class and elucidate the direction of evolution.

new zealand journal of forestry science

The occurrence of progesterone 5β-reductase is not limited to the angiosperms: a functional gene was identified in Picea sitchensis and expressed in Escherichia coli