Evolution of plant secondary metabolism: The promiscuity of short chain dehydrogenases/reductases involved in tropinone reduction
Zusammenfassung der Projektergebnisse
The scope of the joint project was the investigation of the catalytic capacity of tropinone reductase-like enzymes from Arabidopsis thaliana, Capsella rubella in comparison to tropinone reductases of Cochlearia species (all Brassicaceae). The project was a collaboration between the group B. Dräger perfoming the in vitro experiments and the group of W. Brandt contributing theoretical investigations. In Arabidopsis thaliana and Capsella rubella, several genes code for short-chain dehydrogenase/reductases (SDR), with sequences showing a high identity to known tropinone reductases (TRs) from Solanaceae. A. thaliana and C. rubella, other than Cochlearia species, do not form alkaloids of the tropane type. On this account the corresponding enzymes have been classified as tropinone reductase-like enzymes (TRLs), assuming that they reduce carbonyl compounds with NAD(P)H as cofactors. The central question of the project was whether and how TRLs reduce tropinone and which other compounds they accept as substrates. General insights into the evolution of tropane alkaloid biosynthetic enzymes were expected to be derivable. Substrates of TRLs were predicted based on in silico protein structure models and in silico screening performed by the PhD student Juliane Fischer. Potential substrates were then tested by in vitro enzyme measurements performed by the PhD student Nicole Reinhardt. In the beginning of the project, only amino acid sequences and no functional information was available for all enzymes under investagtion. Eight selected TRLs were tested for their ability to use tropanes as substrates. None of them reduced tropinone or oxidized corresponding alcohols in vitro. The experimental results were followed by high quality structural models comparing TRLs with proven TRs. In the TRLs substrate binding sites, a negatively charged amino (Glu or Asp) is missing that binds to the tropane alkaloid skeleton in TRs. Despite of several attempts no crystals of TRLs useful for X-ray structure analysis were obtained. Thus the enzyme models served for ligand identification by in silico pharmacophore search and subsequent docking studies. All Arabidopsis TRLs reduce small lipophilic ketones and lipophilic monoterpene ketones in vitro as it was predicted from their proteins models in silico. Stereospecificity and stereoselectivity in the reduction of monoterpene ketones appear attractive for biotechnological applications. Monoterpenes are widely used as additives for food, cosmetics, or pharmaceutical products. TRL can form pure enantiomers of (-)-thujol and (-)-neothujol, which are not commercially available at present. Equally, (-)-trans-carveol is available only as mixture of cis and trans diastereomers in an unknown ratio. Mutations for faster and more specific reactions and the construction of chimeric TRL are still ongoing experiments. TRLs provide new options for enantio-specific synthesis of oxidized terpenes and further natural compounds. After cloning into microorganisms, TRL capacities can be utilized either in the whole cells or as isolated enzymes. The outcome of the project enables insights into the promiscuity of TRLs as prerequisite for evolution of specific functions in natural product biosynthesis and opens chances for industrial applications of these enzymes in green biotechnology.
Projektbezogene Publikationen (Auswahl)
- 2008. In silico screening and in vitro evaluation of ligands for tropinone reductase-like short-chain dehydrogenases/reductases from Arabidopsis thaliana and Capsella rubella. 6th Kurt Mothes Workshop – Secondary Metabolism, Jena
Reinhardt, N., Navarro-Quezada, A., Schmid, K., Brock, B., Brandt, W., Dräger, B.
- 2009. Investigating biochemical role of tropinone reductase-like short-chain dehydrogenases/reductases for Arabidopsis thaliana and Capsella rubella. Botanikertagung – Plants for the Future, Leipzig
Reinhardt, N., Navarro-Quezada, A., Schmid, K., Fischer, J., Brandt, W., Dräger, B.
- 2009. Tropinone reductase-like short-chain dehydrogenases/reductases in Arabidopsis thaliana and other Brassicaceae. IPMB, St. Louis (USA)
Reinhardt, N., Fischer, J., Brandt, W., Dräger, B.
- 2010. Modelling of short-chain dehydrogenases/reductases and in silico screening for potential ligands. EuroQSAR, Rhodos (GR)
Fischer, J., Reinhardt, N., Dräger, B., Brandt, W., Wessjohann, L. A.
- 2011. Modelling of short-chain dehydrogenases/reductases and in silico screening for potential ligands. 7th Plant Science Student Conference, Halle
Fischer, J., Reinhardt, N., Dräger, B., Brandt, W., Wessjohann, L.
- 2011. Promiscuous but straight: tropinone reductaselike short-chain dehydrogenases/reductases in Brassicaceae. Botanikertagung – Diversity makes the difference, Berlin
Reinhardt, N., Fischer, J., Brandt, W., Dräger, B.
- 2011. Swiss knife enzymes: short chain dehydrogenases/reductases (SDR) with relaxed substrate acceptance are encoded in many in plant genomes. Botanikertagung – Diversity makes the difference, Berlin
Reinhardt, N., Fischer, J., Brandt, W., Dräger, B.
- 2014. Substrate flexibility and reaction specificity of tropinone reductase-like short-chain dehydrogenases. Bioorganic Chemistry 53:37- 49
Reinhardt, N., Fischer, J., Coppi, R., Blum, E., Brandt, W., Dräger, B.
(Siehe online unter https://doi.org/10.1016/j.bioorg.2014.01.004)
