Abstract
Progesterone 5β-reductases (P5sRs) 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 P5sRs 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. A tissue complementary DNA (cDNA) library was screened for orthologous P5sRs. Candidates were subcloned into expression vectors and overexpressed in E. coli cells. The recombinant P5sR 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 P5sR structure of Digitalis lanata and other plant P5sR models. Performing 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.) Carriere putatively encoding P5sRs. Succeeding with the expression of one clone in E. coli, the highly purified protein was unambiguously able to enantioselectively convert progesterone into 5s-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. (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 P5sR gene appears to have existed in the last common ancestor of seed plants approximately 300 million years ago. If the diversification of P5sRs, including the currently detected homologous iridoid synthase activity, was related to stress encountered during the transition to growth on land, then investigation of P5sRs from pteridophytes and bryophytes should improve our knowledge of this enzyme class and elucidate the direction of evolution.
Highlights
Progesterone 5β-reductases (P5ßRs) catalyse one step in the stereospecific biosynthesis of cardenolides
A multiple alignment was carried out by the ClustalW software package maintained on a local computer (Larkin et al 2007) in order to inspect the conifer sequences for the presence of the conserved protein motifs characterizing plant Progesterone 5ß-reductase (P5ßR) proteins
The previously determined crystal structure PDB ID: 2V6G (Thorn et al 2008) of a recombinant P5ßR protein from D. lanata in a complex with the co-substrate NADPH served as the starting structure to make the model
Summary
Protein similarity searches and in silico analyses Current releases of non-redundant databases were screened by the protein blast algorithm operating at the public NCBI server (www.ncbi.nlm.nih.gov/Blastp) to search for P5ßR sequences in conifers. The resulting plasmid (pET28 MSUMO3-ABK24388.1) expresses a hybrid protein under the control of the T7 promoter with an N-terminus of six histidine residues fused in-frame with the ubiquitinrelated SUMO protein as well as the spruce protein This N-terminal modification enhances the solubility of the recombinant protein in E. coli (Butt et al 2005) and aids in its chromatographic separation (see below). Recombinant protein production and purification Escherichia coli strain BL21DE3/pLysS, harbouring the plasmid pET28 M-SUMO3-ABK24388.1, was grown at 30 °C under rigorous shaking (250 rpm) in 1 to 2 L of LB medium supplemented with kanamycin (20 mg/L) and chloramphenicol (10 mg/L). Enzymatic tests, including product identification Enzymes, substrates, co-substrates, and reference substances were provided by Sigma-Aldrich GmbH (Taufkirchen, Germany), except the recombinant P5ßR proteins produced by Munkert et al (2011), Bauer et al (2012), or reported here. Kinetic parameters were obtained from the Shimadzu UV Probe software package
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