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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.

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The multiproduct sesquiterpene synthase MtTPS5 from Medicago truncatula catalyzes the conversion of farnesyl diphosphate (FDP) into a complex mixture of 27 terpenoids. 3-Bromo substrate analogues of geranyl diphosphate (3-BrGDP) and farnesyl diphosphate (3-BrFDP) were evaluated as substrates of MTPS5 enzyme. Kinetic studies demonstrated that these compounds were highly potent competitive inhibitors of the MtTPS5 enzyme with fast binding and slow reversibility. Since there is a lack of knowledge about the crystal structure of multiproduct terpene synthases, these molecules might be ideal candidates for obtaining a co-crystal structure with multiproduct terpene synthases. Due to the structural and mechanistic similarity between various terpene synthases we expect these 3-bromo isoprenoids to be ideal probes for crystal structure studies.

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