Research Grants

Award Year 2005

WERCK-REICHHART Danièle
MØLLER Birger Lindberg
ROEPSTORFF Peter
SLIGAR Stephen
Metabolons and complexity in plant secondary metabolism studied by BIA-MS and nanodisc anchoring

Public abstract Plants synthesize a vast number of secondary metabolites. The diversity of the plant metabolome is further expanded by synthesis of often toxic defense compounds as specific responses to abiotic and biotic environmental stresses. Cytochromes P450 and glycosyl or acyl transferases are key enzyme families involved in their biosynthetic pathways. The number of genes in these families, available to orchestrate the complexity of secondary metabolite synthesis, appears too limited unless the function of the enzymes involved is further modulated by protein-protein interactions at the molecular level. Combinatorially determined substrate specificity of metabolons could be one such mechanism. Metabolons are fragile supramolecular enzyme complexes, difficult to isolate and characterize. However, we have gained biochemical evidence for the involvement of metabolons in the synthesis of phenolic compounds and cyanogenic glucosides. These two well-defined biological systems encompass membrane bound P450s and soluble transferases, and offer a unique possibility to use a series of new biophysical approaches to elucidate how metabolon formation between membrane and soluble enzymes is accomplished at the molecular level. A key issue of these approaches is to preserve stability and proper folding of the P450s which most likely anchor metabolons in the plant membranes, and would behave as insoluble and unstable proteins in an acquous environment. P450 intergrity will be preserved by detergent assisted self-assembly of the heterologously expressed P450 and P450 reductase into nanostructures. After verification of catalytic function and desired stoichiometry of the inserted components, the ability of the P450complexes to engage in macromolecular binding events and metabolon formation will be investigated by surface plasmon resonance spectrometry combined with mass spectrometric analysis. Protein co-localization and interaction will be confirmed in vivo, in yeast and in planta. Understanding optimized assembly of metabolons offers new routes to efficient synthesis of natural products in microorganisms and in planta. This has great industrial implications since many such compounds are expensive and in high demand e.g. because they are valuable medicinal drugs and only available in low amounts or from exotic plants.