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Glucosinolates (GLs) are ß-thioglucoside Nhydroxysulfates found in the angiosperm plant families, mainly present in the order Brassicales. The myrosinase enzyme catalyzes the hydrolysis of the thioglucosidic bond in the GL structure, consequently, an unstable aglucone moiety is released. Spontaneous rearrangement of the aglucone yields isothiocyanates that are toxic to many organisms. Brassicaceae species have specifier proteins that promote aglucone to form different groups of substances such as simple nitriles, epithionitriles and organic thiocyanates. The generation of epithionitriles and organic thiocyanates requires the presence of epithiospecifier protein (ESP) and thiocyanate-forming protein (TFP), respectively. Nitrile-specifier proteins (NSPs) promote the simple nitrile formation. The aim of this work was to make a detailed bioinformatics analysis of NSPs found in the family of Brassicaceae. NSP open reading frames (ORF) in Brassicaceae genomes were scanned in silico. Thus, we created a dataset containing 57 predicted NSPs from different species of Brassicaceae family. Computational analysis revealed that Brassicaceae NSPs were acidic and stability and solubility were high. It has been s een that NSPs may found in different chromosomes in varying numbers in the genome. Finally, phylogenetic analysis of Brassicaceae NSPs showed similarities with the major lineages reported in previous phylogenetic studies of Brassicaceae. [ABSTRACT FROM AUTHOR]

While the defensive function of glucosinolates is well established, their possible role as a nutrient reservoir is poorly understood and glucosinolate turnover pathways have not been elucidated. Previous research showed that glucosinolate content in germinating seeds of Arabidopsis thaliana Columbia-0 (Col-0) increases within the first two to four days on culture medium and then decreases below the level at day 0. In this study we used previously characterized T-DNA mutants to investigate if enzymes known to be involved in glucosinolate breakdown upon tissue damage affect the time course of glucosinolate content in germinating seeds. Besides dormant seeds, we analyzed seeds subjected to stratification in water for up to 72 h or germination on plates for up to ten days. Although seeds of tgg1 tgg2 (deficient in above-ground classical myrosinases) had higher glucosinolate levels than Col-0, the changes during germination were not different to those in seeds of Col-0. This demonstrates that TGG1/TGG2 are not responsible for the decline in glucosinolate content upon germination and suggests the involvement of other enzymes. Expression data extracted from publically available databases show a number of β-glucosidases of the BGLU18–BGLU33 clade to be expressed at specific time points of seed maturation and germination identifying them as good candidates for a role in glucosinolate turnover. Although nitrile-specifier proteins (NSPs) act downstream of myrosinases upon glucosinolate breakdown in tissue homogenates, mutants deficient in either seed-expressed NSP2 or seedling-expressed NSP1 were affected in glucosinolate content in seeds and during stratification or germination when compared to Col-0 indicating a direct role in turnover. The mutant lines nsp1-1, nsp2-1 and nsp2-2 had significantly higher glucosinolate levels in dry seeds than Col-0. After 24 h of stratification in water, nsp2-2 seeds contained 2.3 fold higher levels of glucosinolate than Col-0 seeds. This might indicate downregulation of hydrolytic enzymes when nitrile formation following glucosinolate hydrolysis is impaired. The time course of total glucosinolate content during ten days of germination depended on functional NSP1. Based on the present data, we propose a number of experiments that might aid in establishing the pathway(s) of glucosinolate turnover in germinating A. thaliana seeds.

While the defensive function of glucosinolates is well established, their possible role as a nutrient reservoir is poorly understood and glucosinolate turnover pathways have not been elucidated. Previous research showed that glucosinolate content in germinating seeds of Arabidopsis thaliana Columbia-0 (Col-0) increases within the first two to four days on culture medium and then decreases below the level at day 0. In this study we used previously characterized T-DNA mutants to investigate if enzymes known to be involved in glucosinolate breakdown upon tissue damage affect the time course of glucosinolate content in germinating seeds. Besides dormant seeds, we analyzed seeds subjected to stratification in water for up to 72 h or germination on plates for up to ten days. Although seeds of tgg1 tgg2 (deficient in above-ground classical myrosinases) had higher glucosinolate levels than Col-0, the changes during germination were not different to those in seeds of Col-0. This demonstrates that TGG1/TGG2 are not responsible for the decline in glucosinolate content upon germination and suggests the involvement of other enzymes. Expression data extracted from publically available databases show a number of β-glucosidases of the BGLU18–BGLU33 clade to be expressed at specific time points of seed maturation and germination identifying them as good candidates for a role in glucosinolate turnover. Although nitrile-specifier proteins (NSPs) act downstream of myrosinases upon glucosinolate breakdown in tissue homogenates, mutants deficient in either seed-expressed NSP2 or seedling-expressed NSP1 were affected in glucosinolate content in seeds and during stratification or germination when compared to Col-0 indicating a direct role in turnover. The mutant lines nsp1-1 , nsp2-1 and nsp2-2 had significantly higher glucosinolate levels in dry seeds than Col-0. After 24 h of stratification in water, nsp2-2 seeds contained 2.3 fold higher levels of glucosinolate than Col-0 seeds. This might indicate downregulation of hydrolytic enzymes when nitrile formation following glucosinolate hydrolysis is impaired. The time course of total glucosinolate content during ten days of germination depended on functional NSP1. Based on the present data, we propose a number of experiments that might aid in establishing the pathway(s) of glucosinolate turnover in germinating A. thaliana seeds. [ABSTRACT FROM AUTHOR]

Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets.

As important components of the glucosinolate-myrosinase system, specifier proteins mediate plant defense against herbivory and pathogen attacks. After tissue disruption, glucosinolates are hydrolyzed by myrosinases to instable aglucones, which will rearrange to form defensive isothiocyanates. Nevertheless, this reaction could be redirected to form other products by specifier proteins. Up to now, identified specifier proteins include epithiospecifier proteins (ESPs), thiocyanate forming proteins (TFPs), and nitrile-specifier proteins (NSPs). Recently, the structures of ESP and TFP have been reported. However, both the structure and the catalytic mechanism of NSPs remain enigmatic. Here, we solved the crystal structure of the NSP1 protein from Arabidopsis thaliana (AtNSP1). Structural comparisons with ESP and TFP proteins revealed several structural features of AtNSP1 different from those of the two proteins. Subsequent molecular docking studies showed that the R292 residue in AtNSP1 displayed a conformation different from those of the corresponding residues in ESP and TFP proteins, which might account for the product specificity and catalytic mechanism of AtNSP1. Taken together, the present study provides important insights into the molecular mechanisms underlying the different product spectrums between NSPs and the other two types of specifier proteins, and shed light on the future studies of the detailed mechanisms of other specifier proteins. [ABSTRACT FROM AUTHOR]

One of the best-studied plant defense systems, the glucosinolate-myrosinase system of the Brassicales, is composed of thioglucosides known as glucosinolates and their hydrolytic enzymes, the myrosinases. Tissue disruption brings these components together, and bioactive products are formed as a consequence of myrosinase-catalyzed glucosinolate hydrolysis. Among these products, isothiocyanates have attracted most interest as chemical plant defenses against herbivores and pathogens and health-promoting compounds in the human diet. Previous research has identified specifier proteins whose presence results in the formation of alternative product types, e.g. nitriles, at the expense of isothiocyanates. The biological roles of specifier proteins and alternative breakdown products are poorly understood. Here, we assessed glucosinolate breakdown product profiles obtained upon maceration of roots, seedlings and seeds of Arabidopsis thaliana Columbia-0. We identified simple nitriles as the predominant breakdown products of the major endogenous aliphatic glucosinolates in root, seed, and seedling homogenates. In agreement with this finding, genes encoding nitrile-specifier proteins (NSPs) are expressed in roots, seeds, and seedlings. Analysis of glucosinolate breakdown in mutants with T-DNA insertions in any of the five NSP genes demonstrated, that simple nitrile formation upon tissue disruption depended almost entirely on NSP2 in seeds and mainly on NSP1 in seedlings. In roots, about 70-80 % of the nitrile-forming activity was due to NSP1 and NSP3. Thus, glucosinolate breakdown product profiles are organ-specifically regulated in A. thaliana Col 0, and high proportions of simple nitriles are formed in some parts of the plant. This should be considered in future studies on biological roles of the glucosinolate-myrosinase system.

One of the best-studied plant defense systems, the glucosinolate-myrosinase system of the Brassicales, is composed of thioglucosides known as glucosinolates and their hydrolytic enzymes, the myrosinases. Tissue disruption brings these components together, and bioactive products are formed as a consequence of myrosinase-catalyzed glucosinolate hydrolysis. Among these products, isothiocyanates have attracted most interest as chemical plant defenses against herbivores and pathogens and healthpromoting compounds in the human diet. Previous research has identified specifier proteins whose presence results in the formation of alternative product types, e.g., nitriles, at the expense of isothiocyanates. The biological roles of specifier proteins and alternative breakdown products are poorly understood. Here, we assessed glucosinolate breakdown product profiles obtained upon maceration of roots, seedlings and seeds of Arabidopsis thaliana Columbia-0. We identified simple nitriles as the predominant breakdown products of the major endogenous aliphatic glucosinolates in root, seed, and seedling homogenates. In agreement with this finding, genes encoding nitrile-specifier proteins (NSPs) are expressed in roots, seeds, and seedlings. Analysis of glucosinolate breakdown in mutants with T-DNA insertions in any of the five NSP genes demonstrated, that simple nitrile formation upon tissue disruption depended almost entirely on NSP2 in seeds and mainly on NSP1 in seedlings. In roots, about 70-80% of the nitrile-forming activity was due to NSP1 and NSP3. Thus, glucosinolate breakdown product profiles are organ-specifically regulated in A. thaliana Col-0, and high proportions of simple nitriles are formed in some parts of the plant. This should be considered in future studies on biological roles of the glucosinolate-myrosinase system. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the metabolism of two chain elongated phenolic glucosinolates and the corresponding O-methyl derivatives upon ingestion by caterpillars of the butterfly Pieris rapae (L.). The glucosinolates (GSLs) were 4-hydroxyphenethylGSL, (R)-2-hydroxy-2-(4-hydroxyphenyl)ethylGSL, 4-methoxyphenethylGSL, and (R)-2-hydroxy-2-(4-methoxyphenyl)ethylGSL, variously occurring in foliage of two Arabis species: Arabis hirsuta (L.) Scop. and Arabis soyeri Reut. & Huet subsp. subcoriacea (Gren. ex Nyman) Breitstr. (Brassicaceae). Frass from caterpillars reared on each Arabis species contained two sulfated nitriles (4-sulfates of 3-(4-hydroxyphenyl)propanenitrile and 3-hydroxy-3-(4-hydroxyphenyl)propanenitrile) as apparent GSL metabolites. Comparison of glucosinolate levels in foliage and levels of sulfated nitriles in frass, and experiments with isolated GSLs spiked to crucifer foliage and ingested by P. rapae, demonstrated that phenolic GSLs and the corresponding O-methyl derivatives were metabolised to sulfated nitriles, and that metabolites lacking a β-hydroxy group were partially hydroxylated in this position during metabolism in P. rapae. In contrast, an induction experiment did not show increased levels of β-hydroxylated GSLs in A. soyeri plants upon caterpillar feeding. Frass contents of other putative GSL metabolites from the interaction with the two Arabis species differed significantly; caterpillars reared on A. hirsuta excreted significant amounts of four carboxylic acids (3-(4-hydroxyphenyl)propanoic acid, 3-hydroxy-3-(4-hydroxyphenyl)propanoic acid, and the corresponding 4-sulfates), which were low or absent when the caterpillars were reared on A. soyeri. The excreted carboxylic acids could be formed by hydrolysis of nitriles to carboxylic acids in caterpillar guts by an ingested nitrilase enzyme from A. hirsuta foliage; this hypothesis was supported by demonstration of 3-(4-hydroxyphenyl)propanenitrile hydrolysing nitrilase activity (E.C. 3.5.5.x) in a crude A. hirsuta extract. Some hypothetic metabolites, glycine conjugates of phenolic carboxylic acids, were not detected. Conditions for group separation and HPLC isolation of intact GSLs and sulfated metabolites were optimised, NMR spectroscopic data of the compounds are reported, and evolutionary and ecological implications are discussed. [Copyright &y& Elsevier]

Abstract: A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP–cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation. [Copyright &y& Elsevier]

Glucosinolates are a group of secondary plant metabolites found in the Brassicales order that are beneficial components of our diet, determine the flavor of a number of vegetables and spices and have been implicated in pest management strategies. These properties, most of the biological activities and the pungent odor and taste associated with glucosinolate-containing plants are due to the products formed from glucosinolates by their hydrolytic enzymes, myrosinases, upon tissue disruption. Specifier proteins impact the outcome of glucosinolate hydrolysis without having hydrolytic activity on glucosinolates themselves. In the presence of specifier proteins, glucosinolate hydrolysis results in nitriles, epithionitriles and organic thiocyanates whose biological functions are currently unknown. In contrast, isothiocyanates formed in the absence of specifier proteins have been demonstrated to possess a variety of biological activities and are thought to protect plants from herbivore and pathogen attack. This review discusses the current knowledge on plant and insect specifier proteins with special emphasis on their biochemical properties and possible mechanisms of action. [ABSTRACT FROM AUTHOR]

The defensive function of the glucosinolate–myrosinase system in plants of the order Capparales results from the formation of isothiocyanates when glucosinolates are hydrolysed by myrosinases upon tissue damage. In some glucosinolate-containing plant species, as well as in the insect herbivore Pieris rapae, protein factors alter the outcome of myrosinase-catalysed glucosinolate hydrolysis, leading to the formation of products other than isothiocyanates. To date, two such proteins have been identified at the molecular level, the epithiospecifier protein (ESP) from Arabidopsis thaliana and the nitrile-specifier protein (NSP) from P. rapae. These proteins share no sequence similarity although they both promote the formation of nitriles. To understand the biochemical bases of nitrile formation, we compared some of the properties of these proteins using purified preparations. We show that both proteins appear to be true enzymes rather than allosteric cofactors of myrosinases, based on their substrate and product specificities and the fact that the proportion of glucosinolates hydrolysed to nitriles does not remain constant when myrosinase activity varies. No stable association between ESP and myrosinase could be demonstrated during affinity chromatography, nevertheless some proximity of ESP to myrosinase is required for epithionitrile formation to occur, as evidenced by the lack of ESP activity when it was spatially separated from myrosinase in a dialysis chamber. The significant difference in substrate- and product specificities between A. thaliana ESP and P. rapae NSP is consonant with their different ecological functions. Furthermore, ESP and NSP differ remarkably in their requirements for metal ion cofactors. We found no indications of the involvement of a free radical mechanism in epithionitrile formation by ESP as suggested in earlier reports. [ABSTRACT FROM AUTHOR]

Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets. [ABSTRACT FROM AUTHOR]

Background: Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of approximately 100 amino acids, but the fold of the protein and its biological function are unknown., Objective: We sought to determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays., Methods: nBla g 1 and recombinant constructs were compared by using ELISA with specific murine IgG and human IgE. The structure of Bla g 1 was determined by x-ray crystallography. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to examine the ligand-binding properties of the allergen., Results: The structure of an rBla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by x-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic, and stearic acids were associated with nBla g 1 from cockroach frass. One unit of Bla g 1 was equivalent to 104 ng of allergen., Conclusions: Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with nonspecific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure., (Published by Mosby, Inc.)