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

ABSTRACT: 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 ?-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.

SUBMITTER: Eisenschmidt-Bonn D

PROVIDER: S-EPMC6850609 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Eisenschmidt-Bönn Daniela D Schneegans Nicola N Backenköhler Anita A Wittstock Ute U Brandt Wolfgang W

The Plant journal : for cell and molecular biology 20190429 2

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 t ...[more]

PMID: 30900313

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Project description:Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30-45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.

Project description:Glucosinolates are defense-related secondary metabolites found in Brassicaceae. When Brassicaceae come under attack, glucosinolates are hydrolyzed into different forms of glucosinolate hydrolysis products (GHPs). Among the GHPs, isothiocyanates are the most comprehensively characterized defensive compounds, whereas the functional study of nitriles, another group of GHP, is still limited. Therefore, this study investigates whether 3-butenenitrile (3BN), a nitrile, can trigger the signaling pathways involved in the regulation of defense responses in Arabidopsis thaliana against biotic stresses. Briefly, the methodology is divided into three stages, (i) evaluate the physiological and biochemical effects of exogenous 3BN treatment on Arabidopsis, (ii) determine the metabolites involved in 3BN-mediated defense responses in Arabidopsis, and (iii) assess whether a 3BN treatment can enhance the disease tolerance of Arabidopsis against necrotrophic pathogens. As a result, a 2.5 mM 3BN treatment caused lesion formation in Arabidopsis Columbia (Col-0) plants, a process found to be modulated by nitric oxide (NO). Metabolite profiling revealed an increased production of soluble sugars, Krebs cycle associated carboxylic acids and amino acids in Arabidopsis upon a 2.5 mM 3BN treatment, presumably via NO action. Primary metabolites such as sugars and amino acids are known to be crucial components in modulating plant defense responses. Furthermore, exposure to 2.0 mM 3BN treatment began to increase the production of salicylic acid (SA) and jasmonic acid (JA) phytohormones in Arabidopsis Col-0 plants in the absence of lesion formation. The production of SA and JA in nitrate reductase loss-of function mutant (nia1nia2) plants was also induced by the 3BN treatments, with a greater induction for JA. The SA concentration in nia1nia2 plants was lower than in Col-0 plants, confirming the previously reported role of NO in controlling SA production in Arabidopsis. A 2.0 mM 3BN treatment prior to pathogen assays effectively alleviated the leaf lesion symptom of Arabidopsis Col-0 plants caused by Pectobacterium carotovorum ssp. carotovorum and Botrytis cinerea and reduced the pathogen growth on leaves. The findings of this study demonstrate that 3BN can elicit defense response pathways in Arabidopsis, which potentially involves a coordinated crosstalk between NO and phytohormone signaling.

Project description:Glucosinolates, a group of sulfur-rich thioglucosides found in plants of the order Brassicales, have attracted a lot of interest as chemical defenses of plants and health promoting substances in human diet. They are accumulated separately from their hydrolyzing enzymes, myrosinases, within the intact plant, but undergo myrosinase-catalyzed hydrolysis upon tissue disruption. This results in various biologically active products, e.g. isothiocyanates, simple nitriles, epithionitriles, and organic thiocyanates. While formation of isothiocyanates proceeds by a spontaneous rearrangement of the glucosinolate aglucone, aglucone conversion to the other products involves specifier proteins under physiological conditions. Specifier proteins appear to act with high specificity, but their exact roles and the structural bases of their specificity are presently unknown. Previous research identified the motif EXXXDXXXH as potential iron binding site required for activity, but crystal structures of recombinant specifier proteins lacked the iron cofactor. Here, we provide experimental evidence for the presence of iron (most likely Fe2+) in purified recombinant thiocyanate-forming protein from Thlaspi arvense (TaTFP) using a Ferene S-based photometric assay as well as Inductively Coupled Plasma-Mass Spectrometry. Iron binding and activity depend on E266, D270, and H274 suggesting a direct interaction of Fe2+ with these residues. Furthermore, we demonstrate presence of iron in epithiospecifier protein and nitrile-specifier protein 3 from Arabidopsis thaliana (AtESP and AtNSP3). We also present a homology model of AtNSP3. In agreement with this model, iron binding and activity of AtNSP3 depend on E386, D390, and H394. The homology model further suggests that the active site of AtNSP3 imposes fewer restrictions to the glucosinolate aglucone conformation than that of TaTFP and AtESP due to its larger size. This may explain why AtNSP3 does not support epithionitrile or thiocyanate formation, which likely requires exact positioning of the aglucone thiolate relative to the side chain.

Project description:BackgroundTo understand the mechanism of glucosinolates (GSs) accumulation in the specific organs, combined analysis of physiological change and transcriptome sequencing were applied in the current study. Taking Chinese kale as material, seeds and silique walls were divided into different stages based on the development of the embryo in seeds and then subjected to GS analysis and transcriptome sequencing.ResultsThe main GS in seeds of Chinese kale were glucoiberin and gluconapin and their content changed with the development of the seed. During the transition of the embryo from torpedo- to the early cotyledonary-embryo stage, the accumulation of GS in the seed was accompanied by the salient decline of GS in the corresponding silique wall. Thus, the seed and corresponding silique wall at these two stages were subjected to transcriptomic sequencing analysis. 135 genes related to GS metabolism were identified, of which 24 genes were transcription factors, 81 genes were related to biosynthetic pathway, 25 genes encoded catabolic enzymes, and 5 genes matched with transporters. The expression of GS biosynthetic genes was detected both in seeds and silique walls. The high expression of FMOGS-OX and AOP2, which is related to the production of gluconapin by side modification, was noted in seeds at both stages. Interestingly, the expression of GS biosynthetic genes was higher in the silique wall compared with that in the seed albeit lower content of GS existed in the silique wall than in the seed. Combined with the higher expression of transporter genes GTRs in silique walls than in seeds, it was proposed that the transportation of GS from the silique wall to the seed is an important source for seed GS accumulation. In addition, genes related to GS degradation expressed abundantly in the seed at the early cotyledonary-embryo stage indicating its potential role in balancing seed GS content.ConclusionsTwo stages including the torpedo-embryo and the early cotyledonary-embryo stage were identified as crucial in GS accumulation during seed development. Moreover, we confirmed the transportation of GS from the silique wall to the seed and proposed possible sidechain modification of GS biosynthesis may exist during seed formation.

Dataset Information

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

Publications

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

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