RESUMO
Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (â¼9.4 and â¼34â¯GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.
Assuntos
Tiocianatos , Thlaspi , Tiocianatos/química , Tiocianatos/metabolismo , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Thlaspi/metabolismo , Ferro/metabolismo , OxirreduçãoRESUMO
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 ß-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 in vitro. Based on these insights, we propose that specifier proteins are catalysts that might be classified as Fe2+ -dependent lyases.
Assuntos
Glucosinolatos/metabolismo , Proteínas de Plantas/química , Tiocianatos/metabolismo , Thlaspi/metabolismo , Simulação por Computador , Ferro/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Nitrilas/metabolismo , Proteínas de Plantas/metabolismo , Estrutura Terciária de ProteínaRESUMO
The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these compounds mainly depend on the activity of either type III polyketide synthase-like 2-pyrone synthases or hydroxylating 2-oxoglutarate dependent dioxygenases. In the present study, the substrate specificity of these enzymes is investigated by a systematic screening using both natural and artificial substrates with the aims of efficiently forming (new) products and understanding the underlying catalytic mechanisms. In this framework, we focused on the in vitro functional characterization of a 2-pyrone synthase Gh2PS2 from Gerbera x hybrida and two dioxygenases AtF6'H1 and AtF6'H2 from Arabidopsis thaliana using a set of twenty aromatic and aliphatic CoA esters as substrates. UHPLC-ESI-HRMSn based analyses of reaction intermediates and products revealed a broad substrate specificity of the enzymes, enabling the facile "green" synthesis of this important class of natural products and derivatives in a one-step/one-pot reaction in aqueous environment without the need for halogenated or metal reagents and protective groups. Using protein modeling and substrate docking we identified amino acid residues that seem to be important for the observed product scope.
Assuntos
Arabidopsis , Coenzima A , Ésteres , Pironas , Pironas/metabolismo , Pironas/química , Ésteres/química , Ésteres/metabolismo , Arabidopsis/enzimologia , Especificidade por Substrato , Coenzima A/metabolismo , Coenzima A/química , Simulação de Acoplamento Molecular , Produtos Biológicos/metabolismo , Produtos Biológicos/química , Dioxigenases/metabolismo , Dioxigenases/químicaRESUMO
The 'biogenetic isoprene rule', formulated in the mid 20th century, predicted that terpenoids are biosynthesized via polymerization of C5 isoprene units. The polymerizing enzymes have been identified to be isoprenyl diphosphate synthases, products of which are catalyzed by terpene synthases (TPSs) to achieve vast structural diversity of terpene skeletons. Irregular terpenes (e.g, C11, C12, C16 and C17) are also frequently observed, and they have presumed to be synthesized by the modification of terpene skeletons. This review highlights the exciting discovery of an additional route to the biosynthesis of irregular terpenes which involves the action of a newly discovered enzyme family of isoprenyl diphosphate methyltransferases (IDMTs). These enzymes methylate, and sometimes cyclize, the classical isoprenyl diphosphate substrates to produce modified, non-canonical substrates for specifically evolved TPSs. So far, this new pathway has been found only in bacteria. Structure and sequence comparisons of the IDMTs strongly indicate a conservation of their active pockets and overall topologies. Some bacterial IDMTs and TPSs appear in small gene clusters, which may facilitate future mining of bacterial genomes for identification of irregular terpene-producing enzymes. The IDMT-TPS route for terpenoid biosynthesis presents another example of nature's ingenuity in creating chemical diversity, particularly terpenoids, for organismal fitness.
Assuntos
Metiltransferases , Terpenos , Bactérias/genética , Metiltransferases/genética , Família MultigênicaRESUMO
Glycation is referred to as the interaction of protein amino and guanidino groups with reducing sugars and carbonyl products of their degradation. Resulting advanced glycation end-products (AGEs) contribute to pathogenesis of diabetes mellitus and neurodegenerative disorders. Upon their intestinal absorption, dietary sugars and α-dicarbonyl compounds interact with blood proteins yielding AGEs. Although the differences in glycation potential of monosaccharides are well characterized, the underlying mechanisms are poorly understood. To address this question, d-glucose, d-fructose and l-ascorbic acid were incubated with human serum albumin (HSA). The sugars and α-dicarbonyl intermediates of their degradation were analyzed in parallel to protein glycation patterns (exemplified with hydroimidazolone modifications of arginine residues and products of their hydrolysis) by bottom-up proteomics and computational chemistry. Glycation of HSA with sugars revealed 9 glyoxal- and 14 methylglyoxal-derived modification sites. Their dynamics was sugar-specific and depended on concentrations of α-dicarbonyls, their formation kinetics, and presence of stabilizing residues in close proximity to the glycation sites.