Structural diversification during glucosinolate breakdown: Mechanisms of thiocyanate, epithionitrile and simple nitrile formation.

Secondary metabolism is characterized by an impressive structural diversity. Here, we have addressed the mechanisms underlying structural diversification upon damage-induced activation of glucosinolates, a group of thioglucosides found in the Brassicales. The classical pathway of glucosinolate activation involves myrosinase-catalyzed hydrolysis and rearrangement of the aglucone to an isothiocyanate. Plants of the Brassicaceae possess specifier proteins, i.e. non-heme iron proteins that promote the formation of alternative products by interfering with this reaction through unknown mechanisms. We have used structural information available for the thiocyanate-forming protein from Thlaspi arvense (TaTFP), to test the impact of loops protruding at one side of its beta-propeller structure on product formation using the allylglucosinolate aglucone as substrate. In silico loop structure sampling and semiempirical quantum mechanical calculations identified a 3L2 loop conformation that enabled the Fe2+ cofactor to interact with the double bond of the allyl side chain. Only this arrangement enabled the formation of allylthiocyanate, a specific product of TaTFP. Simulation of 3,4-epithiobutane nitrile formation, the second known product of TaTFP, required an alternative substrate docking arrangement in which Fe2+ interacts with the aglucone thiolate. In agreement with these results, substitution of 3L2 amino acid residues involved in the conformational change as well as exchange of critical amino acid residues of neighboring loops affected the allylthiocyanate versus epithionitrile proportion obtained upon myrosinase-catalyzed allylglucosinolate hydrolysis in the presence of TaTFP invitro. Based on these insights, we propose that specifier proteins are catalysts that might be classified as Fe2+-dependent lyases. Significance Statement Structural diversification upon glucosinolate breakdown is controlled by specifier proteins, a group of non-heme iron proteins. Our study provides mechanistic insights that identify specifier proteins as Fe2+-dependent C-S/C-C lyases. By contrast with the role of Fe2+ in sulfur abstraction during simple nitrile formation, thiocyanate and epithionitrile formation depends on Fe2+/Fe3+ as a redox partner. Thiocyanate formation by Thlaspi arvense thiocyanate-forming protein requires a conformational change that enables an alternative substrate docking pose.