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Tropinone reductase-like enzymes

The promiscuity of tropinone reductase-like short chain dehydrogenases/reductases

Enzymes with relaxed substrate preferences are considered as primordial proteins that served as basis for the evolution of specific catalysts in higher organisms. In higher plants, numerous short chain dehydrogenases/ reductases (SDR) participate in secondary metabolism. These enzymes mostly possess high substrate and reaction specificity, e.g., tropinone reductases. In most plant genomes known so far and in some prokaryote genomes as well, a group of SDR encoding genes are present that are related to tropinone reductases by DNA sequence similarity of more than 50%. They are annotated as tropinone-reductase-like (TRL) genes, suggesting that plants and bacteria possess the capacity to reduce tropinone. The aim of this project is the analysis of catalytic characteristics and evolution of TRL and the definition of their biological functions in plants.

In one part of this project we synthesize TRL-encoded proteins from Brassicaceae and measure their enzymatic properties, i.e., (co-) substrate turnover and specificity. Substrate choice is guided by protein model and pharmacophor construction and subsequent substrate docking. TRL enzymes mostly do not reduce tropinone. In contrast, they are capable of accepting a wide variety of natural and synthetic substrates, including monoterpenes and steroids. Genes encoding substrate-related SDR are an essential part of plant genomes, potentially providing a reservoir for ongoing evolution of metabolic diversity.

In a second part of the project we examine the function of TRL enzymes in vivo using knock-out-plants from Arabidopsis. Modified gene expression patterns of knock-out and wildtype plants can be verified by RNA extraction and northern blot/qRT-PCR analysis. This allows conclusions about specific signal transduction pathways, which are inhibited or promoted by the not any longer synthesized protein. Furthermore metabolite profiling by chromatographic methods coupled with mass spectrometry helps to find inference on the affected pathways. Besides Arabidopsis we work with a second model plant Solanum tuberosum (Solanaceae). Potato contains different tropinone reductases: TRII to produce a precursor for calystegine biosynthesis, one TRI and several TRLs with unknown functions. Transgenic plants producing reduced amounts of the enzyme (RNAi gene silencing) or overproducing the enzyme (gene overexpression) should enable the elucidation of the biological significance of TRI/TRL. Analysis of changes in protein expression patterns and in metabolite profiles may help to clarify the biological function of those enzymes.

Co-operations:

Leibniz-Institute for Plant Biochemistry Halle:

  • Juliane Fischer and Eva Schulze (PhD-students, AG Brandt)
  • Dr. Sabine Rosahl

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