Dataset Information

Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones.

ABSTRACT: 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.

SUBMITTER: Roman J

PROVIDER: S-EPMC7071048 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones.

Román Juan J González Dorian D Inostroza Mario M Mahn Andrea A

Molecules (Basel, Switzerland) 20200211 4

Glucosinolates are secondary plant metabolites of <i>Brassicaceae</i>. 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 o ...[more]

PMID: 32054008

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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:BackgroundThe glucosinolate-myrosinase system is an activated chemical defense system found in plants of the Brassicales order. Glucosinolates are stored separately from their hydrolytic enzymes, the myrosinases, in plant tissues. Upon tissue damage, e.g. by herbivory, glucosinolates and myrosinases get mixed and glucosinolates are broken down to an array of biologically active compounds of which isothiocyanates are toxic to a wide range of organisms. Specifier proteins occur in some, but not all glucosinolate-containing plants and promote the formation of biologically active non-isothiocyanate products upon myrosinase-catalyzed glucosinolate breakdown.ResultsBased on a phytochemical screening among representatives of the Brassicales order, we selected candidate species for identification of specifier protein cDNAs. We identified ten specifier proteins from a range of species of the Brassicaceae and assigned each of them to one of the three specifier protein types (NSP, nitrile-specifier protein, ESP, epithiospecifier protein, TFP, thiocyanate-forming protein) after heterologous expression in Escherichia coli. Together with nine known specifier proteins and three putative specifier proteins found in databases, we subjected the newly identified specifier proteins to phylogenetic analyses. Specifier proteins formed three major clusters, named AtNSP5-cluster, AtNSP1-cluster, and ESP/TFP cluster. Within the ESP/TFP cluster, specifier proteins grouped according to the Brassicaceae lineage they were identified from. Non-synonymous vs. synonymous substitution rate ratios suggested purifying selection to act on specifier protein genes.ConclusionsAmong specifier proteins, NSPs represent the ancestral activity. The data support a monophyletic origin of ESPs from NSPs. The split between NSPs and ESPs/TFPs happened before the radiation of the core Brassicaceae. Future analyses have to show if TFP activity evolved from ESPs at least twice independently in different Brassicaceae lineages as suggested by the phylogeny. The ability to form non-isothiocyanate products by specifier protein activity may provide plants with a selective advantage. The evolution of specifier proteins in the Brassicaceae demonstrates the plasticity of secondary metabolism within an activated plant defense system.

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: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 ?-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.

Project description:Protein L (PpL) is a bacterial protein which is used in the affinity chromatography stage of the production of monoclonal antibodies because of its ability to form high affinity complexes with the light chains of immunoglobulins. In the present work, the binding interfaces between one domain of PpL and antigen-binding fragments (Fab) have been investigated adopting molecular dynamics with the aim of determining the binding contribution of the residues located at the Fab-PpL interface. Because it is known that PpL binds antibodies through two distinct binding sites with different affinities, simulations were performed for both sites to determine interaction free energies to assess the relative binding contribution of the two sites. Mutational studies were then performed only on the dominant binding site. The binding free energy was evaluated with the molecular mechanics Poisson-Boltzmann surface area (MMPBSA) and umbrella sampling/weighted histogram analysis methods. Key residues for the formation of the dominant binding site complex were identified by means of alanine scanning performed both for the Fab and PpL domains. Residues of the light chain of the antibody that contribute most to binding were found to be located between SER7 and VAL13. Four residues from PpL are important for the stability of the complex: PHE839, LYS840, GLU849, and TYR853. Three residues of PpL that do not contribute to the interaction were mutated to histidine (HIS), which changes its protonation state as a function of pH, to find whether this could allow us to control the binding interaction energy. This can be useful in the elution stage of the affinity chromatography purification of antibodies if PpL is used as a ligand. These residues are GLN835, THR836, and ALA837. Molecular dynamics simulations with both protonated and unprotonated HIS were performed to mimic how changing pH may reflect on protein-ligand interaction energies. The MMPBSA approach was used to evaluate the variation of the affinity of the mutated systems with reference to the wild type. Our results show that these mutations could help in disrupting the complex under acidic conditions without impairing the affinity of PpL for the light chains at higher pHs.

Dataset Information

Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones.

Publications

Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones.

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