Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Seit Februar 2021 bietet Wolfgang Brandt, ehemaliger Leiter der Arbeitsgruppe Computerchemie am IPB, sein Citizen Science-Projekt zur Pilzbestimmung an. Dafür hat er in regelmäßigen Abständen öffentliche Vorträge zur Vielfalt…
Gumz, F.; Krausze, J.; Eisenschmidt, D.; Backenköhler, A.; Barleben, L.; Brandt, W.; Wittstock, U.;The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdownPlant Mol. Biol.8967-81(2015)DOI: 10.1007/s11103-015-0351-9
Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide “top” and a narrower “bottom” opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe2+ cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.
Publikation
Brandt, W.; Backenköhler, A.; Schulze, E.; Plock, A.; Herberg, T.; Roese, E.; Wittstock, U.;Molecular models and mutational analyses of plant specifier proteins suggest active site residues and reaction mechanismPlant Mol. Biol.84173-188(2014)DOI: 10.1007/s11103-013-0126-0
As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism.
Publikation
Burow, M.; Rice, M.; Hause, B.; Gershenzon, J.; Wittstock, U.;Cell- and tissue-specific localization and regulation of the epithiospecifier protein in Arabidopsis thalianaPlant Mol. Biol.64173-185(2007)DOI: 10.1007/s11103-007-9143-1
The glucosinolate-myrosinase system found in plants of the order Brassicales is one of the best studied plant defense systems. Hydrolysis of the physiologically inert glucosinolates by hydrolytic enzymes called myrosinases, which only occurs upon tissue disruption, leads to the formation of biologically active compounds. The chemical nature of the hydrolysis products depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles and simple nitriles at the expense of the corresponding isothiocyanates which are formed through spontaneous rearrangement of the aglucone core structure. While isothiocyanates are toxic to a wide range of organisms, including insects, the ecological significance of nitrile formation and thus the role of ESP in plant-insect interactions is unclear. Here, we identified ESP-expressing cells in various organs and several developmental stages of different Arabidopsis thaliana ecotypes by immunolocalization. In the ecotype Landsberg erecta, ESP was found to be consistently present in the epidermal cells of all aerial parts except the anthers and in S-cells of the stem below the inflorescence. Analyses of ESP expression by quantitative real-time PCR, Western blotting, and ESP activity assays suggest that plants control the outcome of glucosinolate hydrolysis by regulation of ESP at both the transcriptional and the post-transcriptional levels. The localization of ESP in the epidermal cell layers of leaves, stems and reproductive organs supports the hypothesis that this protein has a specific function in defense against herbivores and pathogens.