Critical Review. Tipping the Scales Specifier Proteins in Glucosinolate Hydrolysis

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1 IUBMB Life, 59(12): , December 2007 Critical Review Tipping the Scales Specifier Proteins in Glucosinolate Hydrolysis Ute Wittstock and Meike Burow Institut fu r Pharmazeutische Biologie, Technische Universita t Braunschweig, Braunschweig, Germany Summary 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. IUBMB Life, 59: , 2007 Keywords Glucosinolates; myrosinase; epithiospecifier protein; thiocyanate-forming protein; nitrile-specifier protein; epithionitrile; nitrile; organic thiocyanate; isothiocyanate. Abbreviations ESP, epithiospecifier protein; TFP, thiocyanateforming protein; NSP, nitrile-specifier protein; QTL, Quantitative Trait Locus. INTRODUCTION The first descriptions of specifier proteins date back to the late 1960s when proteins were discovered that had an influence on enzymatic reactions, but did not show catalytic activity themselves. Among these proteins were a-lactalbumin that impacts substrate specificity of lactose synthetase (1), colipase that activates lipase by facilitating its adsorption to lipid layers Received 14 September 2007; accepted 9 October 2007 Address correspondence to: Ute Wittstock, Institut fu r Pharmazeutische Biologie, Technische Universita t Braunschweig, Mendelssohnstr. 1, D Braunschweig, Germany. Tel: þ Fax: þ u.wittstock@tu-bs.de (2, 3), and epithiospecifier protein (ESP) that determines the product specificity of plant b-thioglucosidases called myrosinases (4, 5) and is in the focus of this review. The impact of ESP and related proteins on the final outcome of myrosinase-catalyzed reactions is of high interest for agriculture and medicine. The substrates of myrosinases, a class of amino acid-derived plant thioglucosides known as glucosinolates (Fig. 1), are precursors of a diverse range of hydrolysis products which are beneficial components of our diet, determine the flavor and pungency of a number of vegetables and spices from the Brassicales order such as cabbage, broccoli, horseradish and mustard, and have been implicated in pest management strategies (6, 7). From the plant s perspective, glucosinolate hydrolysis products can serve as chemical defenses against herbivory and pathogen attack (8 10). The formation of these bioactive compounds happens during tissue disruption which releases glucosinolates and myrosinases from their separate storage compartments (11). Both the health-promoting effects of glucosinolates and their toxicity to pathogens and herbivores can be attributed mainly to one type of hydrolysis product, the isothiocyanates (mustard oils) and compounds derived from them (10). Isothiocyanates are formed after myrosinase-catalyzed glucosinolate hydrolysis in the absence of specifier proteins by a spontaneous Lossen rearrangement of the aglycone (12, Fig. 2A). When both myrosinase and specifier proteins are present, alternative hydrolysis products such as simple nitriles, epithionitriles, and organic thiocyanates are formed at the expense of isothiocyanates (Fig. 2B). The function of these compounds is largely unknown, and consequently, the biological role of specifier proteins has remained unclear. In the past decade, the availability of molecular tools and genome sequence information has allowed the molecular identification of ESP and related proteins from plants and insects. Biochemical studies of these proteins (13, 14) have suggested that they may, in fact, be enzymes that convert the primary product of the myrosinase-catalyzed reaction into non-isothiocyanate products. Here, we want to review the ISSN print/issn online Ó 2007 IUBMB DOI: /

2 SPECIFIER PROTEINS IN GLUCOSINOLATE HYDROLYSIS 745 when glucosinolates were hydrolyzed by myrosinase at ph 5 5 (12). Only a few years later, evidence was provided that benzylcyanide (phenylacetonitrile) as well as benzylthiocyanate can be formed from benzylglucosinolate after myrosinase-catalyzed hydrolysis at a broader ph range (ph ) under the influence of a so far unknown protein factor or enzyme (4). The formation of an epithionitrile, a nitrile with a terminal thiirane ring (Fig. 2B), was first discovered in seed meal of Crambe abyssinica (Brassicaceae), but subsequently found in several other species containing alkenylglucosinolates with a terminal double bond (16 18). Figure 1. Examples of glucosinolate structures. The glucosinolate core structure consists of a thioglucose linked to a sulfated aldoxime residue and a variable side chain. (A) allylglucosinolate (¼2-propenylglucosinolate), (B) 4-methylthiobutylglucosinolate, (C) benzylglucosinolate, (D) indol-3- ylmethylglucosinolate. current state of research into specifier proteins with special focus on their mechanisms of action. GLUCOSINOLATE HYDROLYSIS PRODUCTS OTHER THAN ISOTHIOCYANATES One of the first reports on the occurrence of glucosinolate hydrolysis products other than isothiocyanates was published in 1959 when benzylthiocyanate and allylthiocyanate were identified as the substances that are responsible for the garliclike odor of Lepidium ruderale, L. sativum and Thlaspi arvense (Brassicaceae), respectively (15). The compounds were shown to be derived from myrosinase-catalyzed hydrolysis of benzylglucosinolate and allylglucosinolate and suggested to be formed under the influence of an additional protein. In 1961, nitriles were demonstrated to be formed spontaneously THE EPITHIOSPECIFIER PROTEIN (ESP) In 1973, Tookey succeeded in separating the protein factor responsible for epithionitrile formation in seeds of Crambe abyssinica from myrosinase and named it epithiospecifier protein (ESP) (5). This name referred to the property of ESP not to attack the intact glucosinolate but to alter the course of the myrosinase-catalyzed reaction (Fig. 2B). ESP was described as a relatively labile protein with a molecular weight of kda that could be stabilized by the addition of Fe 2þ. Subsequently, ESP was shown to be present in other species of the Brassicaceae (19). Following purification of ESP from Brassica napus (20, 21) and elucidation of its partial amino acid sequence (20), the first plant specifier protein was cloned from Arabidopsis thaliana ecotype Landsberg erecta (Ler) (Brassicaceae) by QTL mapping (22). The A. thaliana ESP cdna encodes a 37 kda protein (341 amino acids) with 80% amino acid sequence identity with the partial sequence of the B. napus ESP and 45 55% amino acid sequence identity to several A. thaliana myrosinase binding protein-like proteins (22). Recently, an ESP with 77% amino acid sequence identity to the A. thaliana ESP was identified in Brassica oleracea ssp. italica (23). ESPs are predicted to comprise a series of b-sheets known as Kelch-motifs (13, 23). Since Kelch-motifs have been shown to play a role in protein-protein interactions (24), they may be important for the interaction of ESP with myrosinase (13, 23). Although physical contact between ESP and myrosinase is required for epithionitrile formation to occur, both proteins do not form a stable complex (13, 20, 21) (Fig. 3). It is interesting to note, that the interaction between ESP and myrosinase does not appear to be species specific but occurs across species and even kingdom borders in vitro (25, 26). In the presence of myrosinase, both purified recombinant ESP from A. thaliana and purified recombinant ESP from B. oleracea are able to catalyze the conversion of alkenylglucosinolates to epithionitriles as well as the conversion of nonalkenylglucosinolates and indole glucosinolates to simple nitriles lacking the thiirane ring (13, 23, 27). In contrast to these and other studies that used purified proteins or crude E. coli extracts (22), crude extracts of E. coli expressing A. thaliana ESP were devoid of the ability to support simple

3 746 WITTSTOCK AND BUROW Figure 2. Glucosinolate hydrolysis. Glucosinolates are stored separately from their hydrolytic enzymes, the myrosinases, in the intact plant. Tissue damage abolishes this compartmentalization and initiates the enzymatic activation of glucosinolates. In the first step, myrosinases-catalyzed hydrolysis yields glucose and an unstable aglycone. (A) In the absence of specifier proteins, the aglycone rapidly rearranges to an isothiocyanate. (B) By contrast, the formation of epithionitriles, simple nitriles, and organic thiocyanates depends on the chemical nature of the glucosinolate side chain and involves the action of an additional protein under physiological conditions. ESP, epithiospecifier protein; TFP, thiocyanate-forming protein; NSP, nitrile-specifier protein. nitrile formation in a different study (28). A. thaliana ESP has a distinct substrate specificity with only a small impact on the hydrolysis of benzylglucosinolate, a glucosinolate that does not seem to be present in to A. thaliana (13). Epithionitrile formation is strictly dependent on both ESP and iron (5, 13, 21, 23). The activity of purified recombinant A. thaliana ESP is promoted by ferrous and to a lesser extent by ferric ions (13). In contrast, formation of simple nitriles during myrosinase-catalyzed hydrolysis of glucosinolates may occur in the absence of ESP if iron is present (13, 23, 29). ESP activity in A. thaliana is tightly regulated on both transcriptional and post-transcriptional levels (30). This together with natural variation at the ESP locus (22) and the specific localization of ESP in the epidermal cells of all aboveground organs except the anthers (30), suggests that it has a specific ecological function. Based on insect feeding tests with A. thaliana recombinant inbred lines with allelic variation at the ESP locus and transgenic A. thaliana overexpressing the ESP from the Ler ecotype in the Columbia-0 background devoid of functional ESP, a universal role of simple nitriles in direct defense against generalist herbivores seems unlikely (22, 31). As simple nitriles are volatile compounds, one may speculate that they serve as signals in indirect defense responses. Almost nothing is known about the function of epithionitriles. Due to their reactive thiirane ring, their biological activities are likely to be different from those of simple nitriles (10). The rigorous testing of their ecological role awaits future studies. Recently, an additional role of ESP in leaf senescence has been proposed based on the interaction of ESP with the A. thaliana WRKY53 transcription factor (32). THE THIOCYANATE-FORMING PROTEIN (TFP) While the formation of simple nitriles and epithionitriles is widespread within glucosinolate-containing plants, organic thiocyanates are only generated in very few plant species including Coronopus didymus, Eruca sativa, Lepidium ruderale, L. sativum, and Thlaspi arvense (Brassicaceae) (15, 33, 34). Furthermore, only three glucosinolates, namely allylglucosinolate, 4-methylthiobutylglucosinolate, and benzylglucosinolate (Fig. 1, Fig. 2B), have been reported to form organic thiocyanates. As a common structural feature of these glucosinolates, the ability of their side chains to form stable carbocations has been suggested. This may be a prerequisite for thiocyanate formation (35). After attempts to purify the thiocyanate forming factor from plant extracts had failed (15, 34, Burow and Wittstock, unpublished), the first representative of these proteins, the

4 SPECIFIER PROTEINS IN GLUCOSINOLATE HYDROLYSIS 747 Figure 3. ESP-myrosinase interaction. (A) Spatial sparation of ESP and myrosinase results in non-enzymatic conversion of the aglycone intermediate to an isothiocyanate in vitro (13). (B) ESP has been suggested to interact with myrosinase in an allosteric manner (26) leading to conformational changes in the myrosinase active site that modify the proportions of hydrolysis products formed. (C) Recent findings hint to a catalytic function of ESP (13). Close proximity of the active sites of ESP and myrosinase may be required due to the instability of the aglycone released by myrosinase. ESP, epithiospecifier protein; Glc, glucose; NCS, isothiocyanate; CN, nitrile. thiocyanate forming protein (TFP) from L. sativum, was recently identified using a molecular approach (14). The L. sativum TFP cdna encodes a protein of 337 amino acids (37 kda) with 68% and 63% amino acid sequence identity to the ESPs from A. thaliana and B. oleracea ssp. italica, respectively. The similarity between ESPs and TFP suggests the existence of a small family of plant proteins that impacts glucosinolate hydrolysis without having hydrolytic activity on glucosinolates themselves. The most intriguing feature of the L. sativum TFP is its substrate and product specificity (14). When expressed in E. coli, the protein shows TFP as well as ESP activity. Of the three glucosinolates that can form organic thiocyanates, recombinant TFP promotes thiocyanate formation exclusively from benzylglucosinolate, the predominant endogenous glucosinolate in the above ground organs of L. sativum. In addition to benzylthiocyanate, the corresponding nitrile, benzyl cyanide, is also formed. In contrast, recombinant TFP promotes the formation of the epithionitrile derived from allylglucosinolate and the simple nitrile derived from 4- methylthiobutylglucosinolate, but not the formation of thiocyanates from these glucosinolates (14). Similar to the action of known ESPs, epithionitrile, simple nitrile and thiocyanate formation by TFP is promoted by the presence of iron (14, 36). However, the effect of both ferrous and ferric ions in crude preparations of L. sativum seeds is most pronounced for nitrile formation while thiocyanate formation is reduced at higher iron levels (36). TFP activity in L. sativum is highest in flowers, but absent from leaves, siliques and roots while TFP transcript was detected in all of these organs suggesting post-transcriptional regulation of TFP activity (14). The organ-specific regulation may also indicate that thiocyanate formation fulfills a specific function for the plant. THE NITRILE-SPECIFIER PROTEIN (NSP) A recent study on biochemical mechanisms of adaptation of specialist insect herbivores on glucosinolate-containing plants to their host plants led to the identification of a nitrile-specifier protein (NSP) from larvae of the cabbage white butterfly, Pieris rapae (Lepidoptera: Pieridae) (37). Although functionally related to plant ESPs, NSP has no structural similarities to known plant specifier proteins. The

5 748 WITTSTOCK AND BUROW NSP cdna encodes a 73 kda protein (632 amino acids) including 16 amino acids that serve as a signal peptide for excretion of the protein into the gut lumen (37). NSP belongs to a group of insect proteins that have not been functionally characterized, but have been identified as allergens, for example in cockroach feces. Unlike ESP and TFP, NSP does not contain Kelch motifs, but is made up of sequence repeats stretching about 200 amino acids each. Myrosinase-catalyzed hydrolysis of glucosinolates in the presence of NSP leads to the formation of simple nitriles instead of isothiocyanates (Fig. 2B). In contrast to the isothiocyanates (38), nitriles appear not to be harmful to the larvae and are excreted with the feces (37). Consonant with its ecological role to enable larvae to feed on glucosinolate-containing plants, NSP has a broad substrate specificity and is only expressed in the larval gut (37). It promotes the formation of nitriles from aliphatic as well as aromatic glucosinolates with similar efficiencies (13). NSP activity increases slightly in the presence of ferrous, but not ferric ions. It is, however, not strictly dependent on the presence of iron. Interestingly, purified NSP does not promote epithionitrile formation, even in the presence of 0.01 mm ferrous ions (13). THE BIOCHEMICAL ROLE OF SPECIFIER PROTEINS IN GLUCOSINOLATE HYDROLYSIS The name specifier proteins and some initial studies on their role in glucosinolate hydrolysis suggested that these proteins are cofactors of myrosinase that do not possess catalytic activity on their own (5, 26). If specifier proteins interacted with myrosinase in an allosteric way (Fig. 3), a change in myrosinase activity should not alter the ratio of isothiocyanate to nitrile produced during hydrolysis. This was tested in a recent study using purified recombinant A. thaliana ESP and purified native P. rapae NSP together with purified Sinapis alba myrosinase (13). When myrosinase activity was varied using different reaction temperatures or by the addition of L-ascorbate as activator of myrosinase, the absolute but not the relative amounts of epithionitrile and nitrile, formed in the presence of ESP or NSP, respectively, remained more or less constant. This is not in accordance with an allosteric mechanism, but rather with a catalytic role of specifier proteins (Fig. 3). The demonstration of their enzymatic activity is not trivial as their potential substrates, the glucosinolate aglyca, are unstable and can not be used directly in enzyme assays (Fig. 2B), but the pronounced substrate specificities of ESP and TFP speak for a role as catalysts (13, 14, Fig. 3). Assuming that specifier proteins are enzymes, what could their catalytic role be? Our current knowledge on the mechanisms of simple nitrile, epithionitrile and thiocyanate formation is very limited and we can only speculate about the possible roles of specifier proteins in these quite diverse reactions. Hydrolysis product formation is initiated by the action of myrosinase that cleaves the thioglucosidic bond of the glucosinolate substrate. In the absence of specifier proteins, the aglycone (a thiohydroxamate-o-sulfonate) rearranges to a thioamido sulfate followed by release of sulfate in concert with migration of the aglycone side chain to the nitrogen atom resulting in isothiocyanate formation (Lossen rearrangement, Fig. 2A). In order for thiocyanate formation to occur instead, the sulfur atom originating from the thioglucosidic bond must retain its negative charge to attack electrophilic carbon atoms of the side chain (35, Fig. 4A C). In the case of allylthiocyanate formation, this mechanism is supported by labeling experiments showing that the sulfur atom reacts with the terminal carbon atom of the allyl residue (35, 39) (Fig. 4A). A potential role of TFP could be to preserve the negatively charged sulfur atom and to position it in vicinity to the positive charge of the side chain (Fig. 4A C). The different steric conditions during the nucleophilic attack of the aglyca side chains of allyl-, benzyl- and 4-methylthiobutylglucosinolate might explain the observed substrate specificity of the Lepidium sativum TFP (14). Isotopic labeling experiments demonstrated that epithionitrile formation proceeds via an intramolecular transfer of the thioglucosidic sulfur to the terminal double bond of the glucosinolate side chain (40). In contrast to what would be expected for an enzymatic reaction, the addition is not stereospecific and two diastereomers are formed in a 1:1 ratio (13, 16). Although it seems likely that ESP plays a role in abstracting the sulfur atom from the aglycone and positioning it for the reaction with the double bond, there are no studies that have addressed this question. Epithionitrile formation has been postulated to involve a radical mechanism similar to reactions catalyzed by cytochrome P450 monooxygenases (21, Fig. 4D). In this model, a sulfur radical would be produced by electron transfer from a ferrous ion to the sulfur atom which would then attack the terminal double bond resulting in formation of the thiirane ring. However, experimental data do not support this mechanism as exclusion of oxygen and addition of radical scavengers did not affect the absolute or relative amounts of epithionitrile formed from allylglucosinolate by purified recombinant A. thaliana ESP (13). As alternative hypotheses, the reaction of elemental sulfur with the side chain double bond and the formation of a cyclic intermediate have been discussed (41). The fact that L. sativum TFP promotes epithionitrile formation from allylglucosinolate may suggest that epithionitrile formation proceeds through an ionic mechanism as has been proposed for thiocyanate formation. This would, however, raise the question why in this case the nucleophilic attack of the double bond does not lead to thiocyanate, but to epithionitrile formation. The mechanism of ESP/TFP-dependent simple nitrile formation has rarely been discussed in the literature. As simple nitrile formation by specifier proteins is only observed from glucosinolates missing a terminal double bond, one possibility is that the sulfur atom is released as elemental sulfur

6 SPECIFIER PROTEINS IN GLUCOSINOLATE HYDROLYSIS 749 Figure 4. Proposed reaction mechanisms for thiocyanate and epithionitrile formation by specifier proteins. Thiocyanate formation is restricted to allylglucosinolate (A), benzylglucosinolate (B), and 4-methylthiobutylglucosinolate (C) all of which possess side chain R groups with the ability to form stable carbocations (35, 39). The formation of epithionitriles from alkenylglucosinolates (e.g., allylglucosinolate) has been postulated to occur through a radical mechanism (D) (21). For discussion of the proposed mechanisms, see text. Schemes were redrawn from refs. 21, 35, 39.., isotopic label. due to the lack of a suitable acceptor. Elemental sulfur has in fact been detected upon simple nitrile formation (41). Noticeably, simple nitrile formation upon myrosinase-catalyzed hydrolysis is also promoted by the insect NSP, a protein with a completely different primary and secondary structure than ESP and TFP, as well as at high iron or proton concentrations in the absence of specifier proteins. Ferrous ions have been postulated to bind the negatively charged sulfur derived from the thioglucosidic bond and to be sufficient to abstract it from the aglycone for simple nitrile formation (21). CONCLUSIONS AND FUTURE PERSPECTIVES Considering the high amino acid sequence similarity between ESP and TFP, it is quite astonishing that these proteins are involved in fairly different reactions. However, they may fulfill a common function in glucosinolate hydrolysis by binding the sulfur atom derived from the thioglucosidic bond after myrosinase-catalyzed cleavage and to position it for subsequent reactions with the side chain. While the sulfur atom has to be abstracted from the aglycone backbone for epithionitrile formation, it may remain bound to the glucosinolate core structure during thiocyanate formation. Most remarkably, TFP appears to have both the ability to facilitate the nucleophilic attack leading to benzylthiocyanate formation and the ability to bind the sulfur for epithionitrile formation from allylglucosinolate. This leaves us with many open questions as to how hydrolysis product formation works and which role specifier proteins may play in these reactions. With the availability of cdna sequences of ESPs and TFP, it is now possible to analyze their catalytic mechanisms in more detail. Future research will likely identify amino acid residues that determine substrate and product specificities of specifier proteins as well as residues that are involved in their interaction with myrosinase. The availability of a crystal structure of one of these proteins would be extremely helpful for the identification of their biochemical functions. The use of molecular tools will also facilitate studies on the ecological roles of specifier proteins and their reaction products. The recent findings opened an exciting area of research aimed at elucidating how selection pressure exerted by abiotic and biotic stresses to glucosinolate-containing plants led to the evolution of specifier proteins with different biochemical properties.

7 750 WITTSTOCK AND BUROW ACKNOWLEDGEMENTS We apologize to all our colleagues whose contributions could not be cited due to space limitations. REFERENCES 1. Brew, K., Vanaman, T. C., and Hill, R. L. (1968) The role of a- lactalbumin and the A protein in lactose synthetase: a unique mechanism for the control of a biological reaction. Proc. Natl. Acad. Sci. USA 59, Maylie, M. F., Charles, M., Gache, C., and Desnuelle, P. (1971) Isolation and partial identification of a pancreatic colipase. Biochim. Biophys. Acta 229, Brockman, H. (2002) Colipase-induced reorganization of interfaces as a regulator of lipolysis. Coll. Surf. B: Biointerfaces 26, Virtanen, A. I. (1965) Studies on organic sulphur compounds and other labile substances in plants. Phytochemistry 4, Tookey, H. L. (1973) Crambe thioglucoside glucohydrolase (EC ): separation of a protein required for epithiobutane formation. Can. J. Biochem. 51, Fahey, J. W., Zalcmann, A. T., and Talalay, P. (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, Halkier, B. A., and Gershenzon, J. (2006) Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 57, Chew, F. S. (1988) Biological effects of glucosinolates. In: Biologically Active Natural Products (Cutler, H. G., ed.) pp , American Chemical Society, Washington, DC. 9. Rask, L., Andréasson, E., Ekbom, B., Eriksson, S., Pontoppidan, B., and Meijer, J. (2000) Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Mol. Biol. 42, Wittstock, U., Kliebenstein, D. J., Lambrix, V., Reichelt, M., and Gershenzon, J. (2003) Glucosinolate hydrolysis and its impact on generalist and specialist insect herbivores. In: Integrative Phytochemistry: From Ethnobotany to Molecular Ecology (Romeo, J. T., ed.). pp , Elsevier, Amsterdam. 11. Andréasson, E., and Jørgensen, L. B. (2003) Localization of plant myrosinases and glucosinolates. In: Integrative Phytochemistry: From Ethnobotany to Molecular Ecology (Romeo, J. T., ed.) pp , Elsevier, Amsterdam. 12. Ettlinger, M., Dateo, G. P., Harrison, B., Mabry, T. J., and Thompson, C. P. (1961) Vitamin C as a coenzyme: the hydrolysis of mustard oil glucosides. Proc. Natl. Acad. Sci. USA 47, Burow, M., Markert, J., Gershenzon, J., and Wittstock, U. (2006) Comparative biochemical characterization of nitrile-forming proteins from plants and insects that alter myrosinase-catalysed hydrolysis of glucosinolates. FEBS J. 273, Burow, M., Bergner, A., Gershenzon, J., and Wittstock, U. (2007) Glucosinolate hydrolysis in Lepidium sativum Identification of the thiocyanate-forming protein. Plant Mol. Biol. 63, Gmelin, R., and Virtanen, A. I. (1959) A new type of enzymatic cleavage of mustard oil glucosides. Formation of allylthiocyanate in Thlaspi arvense L. and benzylthiocyanate in Lepidium ruderale L. and Lepidium sativum L. Acta Chem. Scand. 13, Daxenbichler, M. E., VanEtten, C. H., and Wolff, I. A. (1968) Diastereomeric episulfides from epi-progoitrin upon autolysis of crambe seed meal. Phytochemistry 7, Kirk, J. T. O., and MacDonald, C. G. (1974) 1-Cyano-3,4- epithiobutane: a major product of glucosinolate hydrolysis in seeds from certain varieties of Brassica campestris. Phytochemistry 13, Cole, R. (1976) Isothiocyanates, nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae. Phytochemistry 15, MacLeod, A. J., and Rossiter, J. T. (1985) The occurrence and activity of epithiospecifier protein in some Cruciferae seeds. Phytochemistry 24, Bernardi, R., Negri, A., Ronchi, S., and Palmieri, S. (2000) Isolation of the epithiospecifier protein from oil-rape (Brassica napus ssp. oleifera) seed and its characterization. FEBS Lett. 467, Foo, H. L., Groenning, L. M., Goodenough, L., Bones, A. M., Danielsen, B. E., Whiting, D. A., and Rossiter, J. T. (2000) Purification and characterisation of epithiospecifier protein from Brassica napus: enzymic intramolecular sulphur addition within alkenyl thiohydroximates derived from alkenyl glucosinolate hydrolysis. FEBS Lett. 468, Lambrix, V., Reichelt, M., Mitchell-Olds, T., Kliebenstein, D. J., and Gershenzon, J. (2001) The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. Plant Cell 13, Matusheski, N. V., Swarup, R., Juvik, J. A., Mithen, R., Bennett, M., and Jeffery, E. H. (2006) Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane. J. Agric. Food Chem. 54, Adams, J., Kelso, R., and Cooley, L. (2000) The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol. 10, Petroski, R. J., and Tookey, H. L. (1982) Interactions of thioglucoside glucohydrolase and epithiospecifier protein of cruciferous plants to form 1-cyanoepithioalkanes. Phytochemistry 21, Petroski, R. J., and Kwolek, W. F. (1985) Interaction of a fungal thioglucoside glucohydrolase and cruciferous plant epithiospecifier protein to form 1-cyanoepithioalkanes: implications of an allosteric mechanism. Phytochemistry 24, Burow, M., Zhang, Z. Y., Ober, J. A., Lambrix, V. M., Wittstock, U., Gershenzon, J., and Kliebenstein, D. J. (2007) ESP and ESM1 mediate indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate in Arabidopsis. Phytochemistry, doi /j.phytochem Zabala, M. D., Grant, M., Bones, A. M., Bennett, R., Lim, Y. S., Kissen, R., and Rossiter, J. T. (2005) Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana. Phytochemistry 66, Tookey, H. L., and Wolff, I. A. (1970) Effect of organic reducing agents and ferrous ion on thioglucosidase activity in Crambe abyssinica seed. Can. J. Biochem. 48, Burow, M., Rice, M., Hause, B., Gershenzon, J., and Wittstock, U. (2007) Cell- and tissue-specific localization and regulation of the epithiospecifier protein in Arabidopsis thaliana. Plant Mol. Biol. 64, Burow, M., Mu ller, R., Gershenzon, J., and Wittstock, U. (2006) Altered glucosinolate hydrolysis in genetically engineered Arabidopsis thaliana and its influence on the larval development of Spodoptera littoralis. J. Chem. Ecol. 32, Miao, Y., and Zentgraf, U. (2007) The antagonist function of Arabidopsis WRKY53 and ESR/ESP in leaf senescence is modulated by the jasmonic and salicylic acid equilibrium. Plant Cell 19, Walker, N. J., and Gray, I. K. (1970) The glucosinolate of land cress (Coronopus didymus) and its enzymic degradation products as precursors of off-flavor in milk a review. J. Agricul. Food Chem. 18, Schlüter, M., and Gmelin, R. (1972) Abnormale enzymatische Spaltung von 4-Methylthiobutylglucosinolat in Frischpflanzen von Eruca sativa. Phytochemistry 11,

8 SPECIFIER PROTEINS IN GLUCOSINOLATE HYDROLYSIS Lu thy, J., and Benn, M. H. (1977) Thiocyanate formation from glucosinolates: a study of the autolysis of allylglucosinolate in Thlaspi arvense L. seed flour extracts. Can. J. Biochem. 55, Hasapis, X., and MacLeod, A. J. (1982) Effects of metal ions on benzylglucosinolate degradation in Lepidium sativum seed autolysates. Phytochemistry 21, Wittstock, U., Agerbirk, N., Stauber, E. J., Olsen, C. E., Hippler, M., Mitchell-Olds, T., Gershenzon, J., and Vogel, H. (2004) Successful herbivore attack due to metabolic diversion of a plant chemical defense. Proc. Natl. Acad. Sci. USA 101, Agrawal, A. A., and Kurashige, N. S. (2003) A role for isothiocyanates in plant resistance against the specialist herbivore Pieris rapae. J. Chem. Ecol. 29, Rossiter, J. T., Pickett, J. A., Bennett, M. H., Bones, A. M., Powell, G., and Cobb, J. (2007) The synthesis and enzymic hydrolysis of (E)-2- [2,3-2 H 2 ]propenyl glucosinolate: confirmation of the rearrangement of the thiohydroximate moiety. Phytochemistry 68, Brocker, E. R., and Benn, M. H. (1983) The intramolecular formation of epithioalkanenitriles from alkenylglucosinolates by Crambe abyssinica seed flour. Phytochemistry 22, Benn, M. (1977) Glucosinolates. Pure Appl. Chem. 49,