Evolution of plant secondary metabolism: The promiscuity of short chain dehydrogenases/reductases involved in tropinone reduction

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The goal of the project is to study short-chain dehydrogenases/reductases (SDR) for catalytic capacities and evolvability and to gain information on their native function. Tropinone reduction is a step in tropane alkaloid biosynthesis performed by SDR. A tropinone reductase from tropane alkaloid-forming Cochlearia officinalis (Brassicaceae) has 16 orthologs in A. thaliana annotated as “putative tropine reductase” due to sequence identity of >50%. However, A. thaliana does not accumulate tropane alkaloids. Numerous tropinone reductaselike genes conserved in other Brassicaceae genomes suggest that those SDR form a reservoir for the evolution of secondary product enzymes. We will investigate catalytic characteristics of tropinone reductase-like SDR from Brassicaceae that form or do not form tropane alkaloids. SDR enzyme models serve for ligand identification by in silico pharmacophore search and comparison with known Brassicaceae metabolites. Promising ligands will be tested in vitro. Thus, metabolic flexibility and catalytic redundancy are defined, and hints for the native function of the SDR can be expected. Tropanes are tested as substrates to distinguish tropane-accepting SDR and residues decisive for tropinone binding. SDR are mutated to achieve or enhance tropinone reduction and test model-guided in vitro enzyme evolution.