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1.
Glucosinolates, amino acid-derived thioglycosides found in plants of the Brassicales order, are one of the best studied classes of plant secondary metabolites. Together with myrosinases and supplementary proteins known as specifier proteins, they form the glucosinolate–myrosinase system that upon tissue damage gives rise to a number of biologically active glucosinolate breakdown products such as isothiocyanates, epithionitriles and organic thiocyanates involved in plant defense. While isothiocyanates are products of the spontaneous rearrangement of the glucosinolate aglycones released by myrosinase, the formation of epithionitriles and organic thiocyanates depends on both myrosinases and specifier proteins. Hydrolysis product profiles of many glucosinolate-containing plant species indicate the presence of specifier proteins, but only few have been identified and characterized biochemically. Here, we report on cDNA cloning, heterologous expression and characterization of TaTFP, a thiocyanate-forming protein (TFP) from Thlaspi arvense L. (Brassicaceae), that is expressed in all plant organs and can be purified in active form after heterologous expression in Escherichia coli. As a special feature, this protein promotes the formation of allylthiocyanate as well as the corresponding epithionitrile upon myrosinase-catalyzed hydrolysis of allylglucosinolate, the major glucosinolate of T. arvense. All other glucosinolates tested are converted to their simple nitriles when hydrolyzed in the presence of TaTFP. Despite its ability to promote allylthiocyanate formation, TaTFP has a higher amino acid sequence similarity to known epithiospecifier proteins (ESPs) than to Lepidium sativum TFP. However, unlike Arabidopsis thaliana ESP, its activity in vitro is not strictly dependent on Fe2+ addition to the assay mixtures. The availability of TaTFP in purified form enables future studies to be aimed at elucidating the structural bases of specifier protein specificities and mechanisms. Furthermore, identification of TaTFP shows that product specificities of specifier proteins can not be predicted based on amino acid sequence similarity and raises interesting questions about specifier protein evolution. 相似文献
2.
3.
The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system 总被引:2,自引:0,他引:2
Glucosinolates are plant secondary metabolites that are hydrolysed by the action of myrosinases into various products (isothiocyanates,
thiocyanates, epithionitriles, nitriles, oxazolidines). Massive hydrolysis of glucosinolates occurs only upon tissue damage
but there is also evidence indicating metabolism of glucosinolates in intact plant tissues. It was originally believed that
the glucosinolate–myrosinase system in intact plants was stable due to a spatial separation of the components. This has been
coined as the ‘mustard oil bomb’ theory. Proteins that form complexes with myrosinases have been described: myrosinase-binding
proteins (MBPs) and myrosinase-associated proteins (MyAPs/ESM). The roles of these proteins and their biological relevance
are not yet completely known. Other proteins of the myrosinase enzyme system are the epithiospecifier protein (ESP) and the
thiocyanate-forming protein (TFP) that divert the glucosinolate hydrolysis from isothiocyanate production to nitrile/epithionitrile
or thiocyanate production. Some glucosinolate hydrolysis products act as plant defence compounds against insects and pathogens
or have beneficial health effects on humans. In this review, we survey and critically assess the available information concerning
the localization, both at the tissular/cellular and subcellular level, of the different components of the myrosinase enzyme
system. Data from the model plant Arabidopsis thaliana is compared to that from other glucosinolate-producing Brassicaceae in order to show common as well as divergent features
of the ‘mustard oil bomb’ among these species. 相似文献
4.
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. 相似文献
5.
The Arabidopsis Epithiospecifier Protein Promotes the Hydrolysis of Glucosinolates to Nitriles and Influences Trichoplusia ni Herbivory 下载免费PDF全文
Virginia Lambrix Michael Reichelt Thomas Mitchell-Olds Daniel J. Kliebenstein Jonathan Gershenzon 《The Plant cell》2001,13(12):2793-2808
Glucosinolates are anionic thioglucosides that have become one of the most frequently studied groups of defensive metabolites in plants. When tissue damage occurs, the thioglucoside linkage is hydrolyzed by enzymes known as myrosinases, resulting in the formation of a variety of products that are active against herbivores and pathogens. In an effort to learn more about the molecular genetic and biochemical regulation of glucosinolate hydrolysis product formation, we analyzed leaf samples of 122 Arabidopsis ecotypes. A distinct polymorphism was observed with all ecotypes producing primarily isothiocyanates or primarily nitriles. The ecotypes Columbia (Col) and Landsberg erecta (Ler) differed in their hydrolysis products; therefore, the Col x Ler recombinant inbred lines were used for mapping the genes controlling this polymorphism. The major quantitative trait locus (QTL) affecting nitrile versus isothiocyanate formation was found very close to a gene encoding a homolog of a Brassica napus epithiospecifier protein (ESP), which causes the formation of epithionitriles instead of isothiocyanates during glucosinolate hydrolysis in the seeds of certain Brassicaceae. The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase, and thus it was more versatile than previously described ESPs. The role of ESP in plant defense is uncertain, because the generalist herbivore Trichoplusia ni (the cabbage looper) was found to feed more readily on nitrile-producing than on isothiocyanate-producing Arabidopsis. However, isothiocyanates are frequently used as recognition cues by specialist herbivores, and so the formation of nitriles instead of isothiocyanates may allow Arabidopsis to be less apparent to specialists. 相似文献
6.
Wolfgang Brandt Anita Backenköhler Eva Schulze Antje Plock Thomas Herberg Elin Roese Ute Wittstock 《Plant molecular biology》2014,84(1-2):173-188
As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism. 相似文献
7.
Zabala Mde T Grant M Bones AM Bennett R Lim YS Kissen R Rossiter JT 《Phytochemistry》2005,66(8):859-867
Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles. 相似文献
8.
9.
ABSTRACT: BACKGROUND: The 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. RESULTS: Based 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, but not within the AtNSP1 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. CONCLUSIONS: Among 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 appearance of lineage I and expanded lineage II of the Brassicaceae. TFP activity evolved from ESPs at least twice independently in different Brassicaceae lineages. 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. 相似文献
10.
The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed. 相似文献
11.
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. 相似文献
12.
Burow M Losansky A Müller R Plock A Kliebenstein DJ Wittstock U 《Plant physiology》2009,149(1):561-574
Glucosinolates are a group of thioglucosides that are components of an activated chemical defense found in the Brassicales. Plant tissue damage results in hydrolysis of glucosinolates by endogenous thioglucosidases known as myrosinases. Spontaneous rearrangement of the aglucone yields reactive isothiocyanates that are toxic to many organisms. In the presence of specifier proteins, alternative products, namely epithionitriles, simple nitriles, and thiocyanates with different biological activities, are formed at the expense of isothiocyanates. Recently, simple nitriles were recognized to serve distinct functions in plant-insect interactions. Here, we show that simple nitrile formation in Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 rosette leaves increases in response to herbivory and that this increase is independent of the known epithiospecifier protein (ESP). We combined phylogenetic analysis, a screen of Arabidopsis mutants, recombinant protein characterization, and expression quantitative trait locus mapping to identify a gene encoding a nitrile-specifier protein (NSP) responsible for constitutive and herbivore-induced simple nitrile formation in Columbia-0 rosette leaves. AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. Four of these homologues possess one or two lectin-like jacalin domains, which share a common ancestry with the jacalin domains of the putative Arabidopsis myrosinase-binding proteins MBP1 and MBP2. A sixth ESP homologue lacked specifier activity and likely represents the ancestor of the gene family with a different biochemical function. By illuminating the genetic and biochemical bases of simple nitrile formation, our study provides new insights into the evolution of metabolic diversity in a complex plant defense system. 相似文献
13.
Genotype, age, tissue, and environment regulate the structural outcome of glucosinolate activation 总被引:5,自引:1,他引:4
Glucosinolates are the inert storage form of a two-part phytochemical defense system in which the enzyme myrosinase generates an unstable intermediate that rapidly rearranges into the biologically active product. This rearrangement step generates simple nitriles, epithionitriles, or isothiocyanates, depending on the structure of the parent glucosinolate and the presence of proteins that promote specific structural outcomes. Glucosinolate accumulation and myrosinase activity differ by plant age and tissue type and respond to environmental stimuli such as planting density and herbivory; however, the influence of these factors on the structural outcome of the rearrangement step remains unknown. We show that the structural outcome of glucosinolate activation is controlled by interactions among plant age, planting density, and natural genetic variation in Arabidopsis (Arabidopsis thaliana) rosette leaves using six well-studied accessions. We identified a similarly complex interaction between tissue type and the natural genetic variation present within these accessions. This raises questions about the relative importance of these novel levels of regulation in the evolution of plant defense. Using mutants in the structural specifier and glucosinolate activation genes identified previously in Arabidopsis rosette leaves, we demonstrate the requirement for additional myrosinases and structural specifiers controlling these processes in the roots and seedlings. Finally, we present evidence for a novel EPITHIOSPECIFIER PROTEIN-independent, simple nitrile-specifying activity that promotes the formation of simple nitriles but not epithionitriles from all glucosinolates tested. 相似文献
14.
15.
ESP and ESM1 mediate indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate in Arabidopsis 总被引:3,自引:0,他引:3
Burow M Zhang ZY Ober JA Lambrix VM Wittstock U Gershenzon J Kliebenstein DJ 《Phytochemistry》2008,69(3):663-671
Glucosinolates are plant secondary metabolites that act as direct defenses against insect herbivores and various pathogens. Recent analysis has shown that methionine-derived glucosinolates are hydrolyzed/activated into either nitriles or isothiocyanates depending upon the plants genotype at multiple loci. While it has been hypothesized that tryptophan-derived glucosinolates can be a source of indole-acetonitriles, it has not been explicitly shown if the same proteins control nitrile production from tryptophan-derived glucosinolates as from methionine-derived glucosinolates. In this report, we formally test if the proteins involved in controlling aliphatic glucosinolate hydrolysis during tissue disruption can control production of nitriles during indolic glucosinolate hydrolysis. We show that myrosinase is not sufficient for indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate and requires the presence of functional epithospecifier protein in planta and in vitro to produce significant levels of indol-3-acetonitrile. This reaction is also controlled by the Epithiospecifier modifier 1 gene. Thus, like formation of nitriles from aliphatic glucosinolates, indol-3-acetonitrile production following tissue disruption is controlled by multiple loci raising the potential for complex regulation and fine tuning of indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate. 相似文献
16.
David J. Williams Christa Critchley Sharon Pun Mridusmita Chaliha Timothy J. OHare 《Phytochemistry》2009,70(11-12):1401-1409
Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress (Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress (Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 °C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 °C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold. 相似文献
17.
Myrosinase: gene family evolution and herbivore defense in Brassicaceae 总被引:33,自引:0,他引:33
Rask L Andréasson E Ekbom B Eriksson S Pontoppidan B Meijer J 《Plant molecular biology》2000,42(1):93-114
Glucosinolates are a category of secondary products present primarily in species of the order Capparales. When tissue is damaged, for example by herbivory, glucosinolates are degraded in a reaction catalyzed by thioglucosidases, denoted myrosinases, also present in these species. Thereby, toxic compounds such as nitriles, isothiocyanates, epithionitriles and thiocyanates are released. The glucosinolate-myrosinase system is generally believed to be part of the plant's defense against insects, and possibly also against pathogens. In this review, the evolution of the system and its impact on the interaction between plants and insects are discussed. Further, data suggesting additional functions in the defense against pathogens and in sulfur metabolism are reviewed. 相似文献
18.
Rohini Bhat 《Critical reviews in biotechnology》2019,39(4):508-523
Glucosinolate–myrosinase is a substrate-enzyme defense mechanism present in Brassica crops. This binary system provides the plant with an efficient system against herbivores and pathogens. For humans, it is well known for its anti-carcinogenic, anti-inflammatory, immunomodulatory, anti-bacterial, cardio-protective, and central nervous system protective activities. Glucosinolate and myrosinase are spatially present in different cells that upon tissue disruption come together and result in the formation of a variety of hydrolysis products with diverse physicochemical and biological properties. The myrosinase-catalyzed reaction starts with cleavage of the thioglucosidic linkage resulting in release of a D-glucose and an unstable thiohydroximate-O-sulfate. The outcome of this thiohydroximate-O-sulfate has been shown to depend on the structure of the glucosinolate side chain, the presence of supplementary proteins known as specifier proteins and/or on the physiochemical condition. Myrosinase was first reported in mustard seed during 1939 as a protein responsible for release of essential oil. Until this date, myrosinases have been characterized from more than 20 species of Brassica, cabbage aphid, and many bacteria residing in the human intestine. All the plant myrosinases are reported to be activated by ascorbic acid while aphid and bacterial myrosinases are found to be either neutral or inhibited. Myrosinase catalyzes hydrolysis of the S-glycosyl bond, O-β glycosyl bond, and O-glycosyl bond. This review summarizes information on myrosinase, an essential component of this binary system, including its structural and molecular properties, mechanism of action, and its regulation and will be beneficial for the research going on the understanding and betterment of the glucosinolate–myrosinase system from an ecological and nutraceutical perspective. 相似文献
19.
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.Brassicaceae plants such as oilseed rape (Brassica napus), turnip
(Brassica rapa), and white mustard (Sinapis alba) as well as
the model plant Arabidopsis thaliana contain a group of secondary
metabolites known as glucosinolates
(GSLs)2
(1,
2). These are
β-thioglucoside N-hydroxysulfates with a sulfur-linked
β-d-glucopyranose moiety and a variable side chain that is
derived from one of eight amino acids or their methylene group-elongated
derivatives. Aliphatic GSLs are derived from alanine, leucine, isoleucine,
valine, or predominantly methionine. Tyrosine or phenylalanine give aromatic
GSLs, and tryptophan-derived GSLs are called indolic GSLs (for review, see
Ref. 3). Although more than 120
different GSLs have been identified in total so far, individual plant species
usually contain only a few GSLs
(2). Quantitative and
qualitative differences of GSL profiles are also observed within a species,
such as, for example, for different A. thaliana ecotypes
(4–6).
In addition, GSL composition varies among organs and during the life cycle of
plants (7,
8) and is affected by external
factors (9).Intact GSLs are mostly considered to be biologically inactive. Most GSL
degradation products have toxic effects on insect, fungal, and bacterial
pests, serve as attractants for specialist insects, or may have beneficial
health effects for humans
(10–15).
The enzymatic degradation of GSLs (Fig.
1A), which occurs massively upon tissue damage, is
catalyzed by plant thioglucosidases called myrosinases (EC 3.2.1.147;
glycoside hydrolase family 1). Depending on several factors (e.g. GSL
structure, proteins, cofactors, pH) myrosinase-catalyzed hydrolysis of GSLs
can lead to a variety of products (Fig.
1B; for review, see Refs.
16 and
17). Of these, isothiocyanates
are the most common as their formation only requires myrosinase activity.
Thiocyanates on the other hand are only produced from a very limited number of
GSLs, and their formation necessitates the presence of a thiocyanate-forming
factor in addition to myrosinase
(18). A thiocyanate-forming
protein (TFP) has recently been identified in Lepidium sativum
(19). Alkenyl GSLs, a subgroup
of aliphatic GSLs containing a terminal unsaturation in their side chain, can
lead to the production of epithionitriles through the cooperative action of
myrosinase and a protein called epithiospecifier protein (ESP
(20)) in a ferrous
ion-dependent way
(21–23).
Both TFP and ESP contain a series of Kelch repeats
(19). Kelch repeats are
involved in protein-protein interactions, and Kelch repeat-containing proteins
are involved in a number of diverse biological processes
(24). In addition to
isothiocyanates, nitriles are the major group of GSL hydrolysis products.
Although ESP and TFP activities can generate nitriles
(19,
21,
25,
26), indications for an
ESP-independent nitrile-specifier activity exist. The GSL hydrolysis profile
of A. thaliana roots, an organ that does not show ESP expression or
activity (27), reveals
predominantly the presence of nitriles
(28). In addition, leaf tissue
of A. thaliana ecotypes supposedly devoid of ESP activity produces a
certain amount of nitriles upon autolysis
(21). Under acidic buffer
conditions, a non-enzymatic production of nitriles from GSLs is observed (Ref.
29 and references therein).
Increasing Fe2+ concentrations have also been shown to favor
nitrile formation over isothiocyanate formation from a number of GSLs in the
presence of myrosinase and absence of ESP
(21,
22). Therefore, a
non-enzymatic origin of this nitrile production cannot be excluded, although
the presence of a nitrile-specifier protein is a tempting alternative.
Although ESP is able to generate nitriles, it has also been shown that the
conversion rates of GSLs to nitriles are lower than those of GSLs to
epithionitriles for ESP (21,
22).Open in a separate windowFIGURE 1.Simplified scheme of enzymatic GSL hydrolysis (A) and
structures and names of GSLs and their hydrolysis products that are mentioned
in the article. (B). A, myrosinase acts on GSLs to form
an unstable aglycone intermediate that can rearrange spontaneously to form an
isothiocyanate. Hydrolysis can be diverted from this default route under
certain conditions (e.g. the presence of NSPs, ferrous ions, or at pH
< 5) to give the corresponding nitrile. ESP is responsible for the
formation of epithionitriles from alkenyl GSLs in a ferrous ion-dependent
mechanism. B, the general structure of GSLs, indicating the variable
side chain as R, is given as well as the three major classes of hydrolysis
products (i.e. isothiocyanates, nitriles, and epithionitriles). The
listed GSLs are the ones mentioned in this article and are arranged according
to the class of GSLs they belong to and with an increase in chain length or
complexity. The names of the respective hydrolysis products are given for a
better understanding of the present article, and not all were encountered
during our studies.A nitrile-specifier protein (NSP) that is able to redirect the hydrolysis
of GSLs toward nitriles has been cloned from the larvae of the butterfly
Pieris rapae (30).
This protein does not, however, exhibit sequence similarity to plant ESP, and
a corresponding plant nitrile-specifier protein has not yet been identified.
We report here the identification of a group of six A. thaliana genes
with some sequence similarity to A. thaliana ESP, providing evidence
for a new family of nitrile-specifier proteins and a more detailed
characterization of one member that possesses nitrile-specifier activity
in vitro, when applied exogenously to plant tissue and after ectopic
expression in the two A. thaliana ecotypes Col-0 and C24. Despite its
sequence homology to A. thaliana epithiospecifier protein (AtESP), it
does not possess epithiospecifier activity under similar conditions.
Therefore, we propose to designate this protein as A. thaliana
nitrile-specifier protein 2 (AtNSP2). Although the biological roles of AtNSP2
and related proteins are not yet known, their specificities and distinctive
expression patterns indicate the presence of a fine-tuned mechanism for GSL
degradation controlling the outcome of an array of biologically active
molecules. 相似文献
20.
Natalia Bellostas Anne D. Srensen Jens C. Srensen Hilmer Srensen 《Journal of Molecular Catalysis .B, Enzymatic》2009,57(1-4):229-236
The ratio of isothiocyanates (ITCs) to nitriles formed in the myrosinase-catalyzed hydrolysis of glucosinolates is a key factor determining the physiological effect of glucosinolate containing plants and materials. In this context, the mechanism by which nitrile formation occurs is not well understood. In the present paper we have studied the effect of three redox reagents – Fe2+, glutathione (GSH) and ascorbic acid – on the profile of products obtained upon the hydrolysis of a model glucosinolate (glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate)) catalyzed by Brassica carinata myrosinase. A Micellar Electrokinetic Capillary Chromatography method that allows following on-line the hydrolysis of the glucosinolate, the formation of the degradation products and the oxidation of GSH was used. Increasing the concentration of Fe2+ and GSH (from 0.25- to 2-fold molar excess with respect to the glucosinolate) increased the ratio of nitrile ((2R)-2-hydroxy-2-phenylethylcyanide) to oxazolidine-2-thione ((5S)-5-phenyloxazolidine-2-thione), whereas increasing the concentration of ascorbic acid decreased this ratio. Low concentrations of ascorbic acid favored nitrile formation. A mechanism for nitrile formation involving a disulfide bond in the myrosinase complex is proposed. 相似文献