BIOLOGY AND BIOCHEMISTRY OF GLUCOSINOLATES
ANRV274-PP57-12ARI27 March 200610:25Annu. Rev. Plant Biol. 2006.57:303-333. Downloaded from arjournals.annualreviews.orgby University of California - Davis on 10/15/08. For personal use only.Biology and Biochemistryof GlucosinolatesBarbara Ann Halkier1 and Jonathan Gershenzon21Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary andAgricultural University, DK-1871 Frederiksberg C, Denmark; email: bah@kvl.dk2Department of Biochemistry, Max Planck Institute for Chemical Ecology,D-07745 Jena, Germany; email: gershenzon@ice.mpg.deAnnu. Rev. Plant Biol.2006. 57:303–33The Annual Review ofPlant Biology is online atplant.annualreviews.orgdoi: 10.1146/annurev.arplant.57.032905.105228c 2006 byCopyright Annual Reviews. All rightsreservedFirst published online as aReview in Advance onJanuary 30, 20061543-5008/06/0602-0303 20.00Key Wordsmetabolic engineering, biosynthesis, degradation, regulation,transport, defenseAbstractGlucosinolates are sulfur-rich, anionic natural products that uponhydrolysis by endogenous thioglucosidases called myrosinases produce several different products (e.g., isothiocyanates, thiocyanates,and nitriles). The hydrolysis products have many different biologicalactivities, e.g., as defense compounds and attractants. For humansthese compounds function as cancer-preventing agents, biopesticides, and flavor compounds. Since the completion of the Arabidopsisgenome, glucosinolate research has made significant progress, resulting in near-complete elucidation of the core biosynthetic pathway, identification of the first regulators of the pathway, metabolicengineering of specific glucosinolate profiles to study function, aswell as identification of evolutionary links to related pathways. Although much has been learned in recent years, much more awaitsdiscovery before we fully understand how and why plants synthesizeglucosinolates. This may enable us to more fully exploit the potentialof these compounds in agriculture and medicine.303
ANRV274-PP57-12ARI27 March 200610:25ContentsAnnu. Rev. Plant Biol. 2006.57:303-333. Downloaded from arjournals.annualreviews.orgby University of California - Davis on 10/15/08. For personal use only.INTRODUCTION . . . . . . . . . . . . . . . . .Chemical Structure and HydrolysisImportance to Humans . . . . . . . . . . .BIOSYNTHESIS . . . . . . . . . . . . . . . . . . .BIOSYNTHESIS: AMINO ACIDCHAIN ELONGATION . . . . . . . .BIOSYNTHESIS: CORESTRUCTURE. . . . . . . . . . . . . . . . . . .The Conversion of Amino Acids toAldoximes . . . . . . . . . . . . . . . . . . . . .The Conversion of Aldoximes toThiohydroximic Acids . . . . . . . . .The Conversion ofThiohydroximic Acids toGlucosinolates . . . . . . . . . . . . . . . .The Evolutionary Link betweenGlucosinolates and CyanogenicGlucosides . . . . . . . . . . . . . . . . . . . .BIOSYNTHESIS: SECONDARYTRANSFORMATIONS . . . . . . . . .REGULATION OFBIOSYNTHESIS . . . . . . . . . . . . . . . .DEGRADATION. . . . . . . . . . . . . . . . . . .Hydrolysis Products . . . . . . . . . . . . . .Biochemistry and Physiologyof Myrosinases . . . . . . . . . . . . . . . .METABOLIC LINKS BETWEENGLUCOSINOLATEMETABOLISM, IAA, ANDOTHER INDOLECOMPOUNDS . . . . . . . . . . . . . . . . .TRANSPORT IN PLANTS . . . . . . . .BIOLOGICAL FUNCTION . . . . . . .METABOLIC ENGINEERINGOF GLUCOSINOLATES . . . . . . .PERSPECTIVE . . . . . . . . . . . . . . . . . . . 17319320322INTRODUCTIONGlucosinolates, once known as mustard oilglucosides, have been part of human life forthousands of years because of the strongflavors and tastes they elicit in cabbage,broccoli, and other Brassica vegetables. Inthe past few decades, the importance ofGlucosinolates:mustard oilglucosides304Halkier·Gershenzonthese nitrogen- and sulfur-containing plantsecondary metabolites has increased further following discovery of their potentialas cancer-prevention agents, crop-protectioncompounds, and biofumigants in agriculture.Moreover, the presence of glucosinolates inthe model plant, Arabidopsis thaliana, has alsohelped to stimulate a vigorous research effort into these unusual amino acid–derivedproducts. For such a widely studied group ofplant compounds, glucosinolates are knownfrom only a few angiosperm families. Theyhave been reported almost exclusively fromthe order Capparales, which contains 15 families, including the Brassicaceae, Capparaceae,and Caricaceae (144). Curiously, glucosinolates are also known from the genus Drypetesof the family Euphorbiaceae, a genus completely unrelated to the other glucosinolatecontaining families.Chemical Structure and HydrolysisThe approximately 120 described glucosinolates share a chemical structure consisting ofa β-D-glucopyranose residue linked via a sulfur atom to a (Z )-N-hydroximinosulfate ester,plus a variable R group (Figure 1) derivedfrom one of eight amino acids (49). Glucosinolates can be classified by their precursoramino acid and the types of modification tothe R group. Compounds derived from Ala,Leu, Ile, Met, or Val are called aliphatic glucosinolates, those derived from Phe or Tyr arecalled aromatic glucosinolates, and those derived from Trp are called indole glucosinolates. The R groups of most glucosinolatesare extensively modified from these precursor amino acids, with methionine undergoingan especially wide range of transformations(49). Most of the R groups are elongated byone or more methylene moieties. Both elongated and nonelongated R groups are subjectto a wide variety of transformations, including hydroxylation, O-methylation, desaturation, glycosylation, and acylation.Plants accumulating glucosinolates always possess a thioglucoside glucohydrolase
Annu. Rev. Plant Biol. 2006.57:303-333. Downloaded from arjournals.annualreviews.orgby University of California - Davis on 10/15/08. For personal use only.ANRV274-PP57-12ARI27 March 200610:25activity known as myrosinase, which hydrolyzes the glucose moiety on the main skeleton (140). The products are glucose and anunstable aglycone that can rearrange to formisothiocyanates, nitriles, and other products.Hydrolysis in intact plants appears to be hindered by the spatial separation of glucosinolates and myrosinase or the inactivation of myrosinase, but these components mix togetherupon tissue damage, leading to the rapid formation of glucosinolate hydrolysis products.Most of the biological activities of glucosinolates are attributed to the actions of theirhydrolysis products (170).GlucosinolatestructureSRGlcNOSO3R AllylglucosinolateBenzylglucosinolateImportance to HumansGlucosinolates have long been of interestto human society because of their presencein certain Brassicaceae vegetables (cabbage,cauliflower, broccoli) and condiments (mustard, horseradish, wasabi). The distinct tasteand flavors of these foods are due primarily totheir isothiocyanate hydrolysis products. Indole glucosinolates and those with alkenyl Rgroups are especially known for causing bitterness (46).In the past 30 years, glucosinolates haveassumed major agricultural significance withthe increasing importance of rapeseeds, cultivars of Brassica napus, B. rapa, and B. juncea,as oil crops in temperate and subtropical areasof the world. These species contain glucosinolates in all of their organs. However, plantbreeders have drastically reduced the levelsof seed glucosinolates to allow the proteinrich seed cake (the residue left after crushingfor oil) to be sold as an animal feed supplement. One of the predominant rapeseed glucosinolates, 2-hydroxy-3-butenyl glucosinolate (Figure 1), forms a oxazolidine-2-thioneupon hydrolysis that causes goiter and hasother harmful effects on animal nutrition (63).Breeders have attempted to modify glucosinolate levels in rapeseed foliage to reduce damage from fungal and insect pests (122). In thiscase, the strategy is not as simple becauseglucosinolates and their hydrolysis ylsulfinylbutylglucosinolateFigure 1Chemical structure of glucosinolates. The common structure is shown, aswell as examples of some specific glucosinolates cited in the text that showtypical variation in the structure of the side chain.are repellent to some insects, but often serveas attractants for others. Brassica cultivars arefinding increased use for “biofumigation,” inwhich harvested plant material is incorporatedinto agricultural soils to suppress pathogens,nematodes, and weeds (22, 164, 174). Hereagain glucosinolate hydrolysis products areassumed to be the active agents of thetreatment.In the past decade, certain glucosinolateshave been identified as potent cancerprevention agents in a wide range of animalmodels due to the ability of certain hydrolysis products to induce phase II detoxification enzymes, such as quinone reductase,glutathione-S-transferase, and glucuronosyl transferases (72b, 81). Sulforaphane, thewww.annualreviews.org GlucosinolatesMyrosinase:β-thioglucosidase305
ARI27 March 200610:25isothiocyanate derivative of 4-methylsulfinylbutyl glucosinolate (Figure 1), found inbroccoli, has been the focus of many ofthese studies (176). Sulforaphane and otherisothiocyanates may prevent tumor growth byblocking the cell cycle and promoting apoptosis (81, 107, 155). Moreover, sulforaphaneexhibits potential for treating Helicobacterpylori-caused gastritis and stomach cancer(48). These results are motivating efforts toincrease the sulforaphane content of broccoliand to promote the health benefits of thisvegetable.Annu. Rev. Plant Biol. 2006.57:303-333. Downloaded from arjournals.annualreviews.orgby University of California - Davis on 10/15/08. For personal use only.ANRV274-PP57-12BIOSYNTHESISThe formation of glucosinolates can be conveniently divided into three separate phases.First, certain aliphatic and aromatic aminoacids are elongated by inserting methylenegroups into their side chains. Second, theamino acid moiety itself, whether elongatedor not, is metabolically reconfigured to givethe core structure of glucosinolates. Third,the initially formed glucosinolates are modified by various secondary transformations.BIOSYNTHESIS: AMINO ACIDCHAIN ELONGATIONThe sequence of the chain-elongation pathway for amino acids participating in glucosinolate biosynthesis is based on in vivofeeding studies, the demonstration of enzymeactivities in vitro, and the isolation of key intermediates. Initially, the parent amino acidis deaminated to form the corresponding 2oxo acid (Figure 2). Next is a three-step cyclein which (1) the 2-oxo acid condenses withacetyl-CoA to form a substituted 2-malatederivative, which then (2) isomerizes via a 1,2hydroxyl shift to a 3-malate derivative that(3) undergoes oxidation-decarboxylation toyield a 2-oxo acid with one more methylenegroup than the starting compound. Duringeach round of the elongation cycle, the twocarbons of acetyl-CoA are added to the 2-oxoacid and the COOH group added in the pre306Halkier·Gershenzonvious round is lost, for a net gain of one carbon atom. After each turn of the cycle, theextended 2-oxo acid can be transaminated toform the corresponding amino acid and enter the second phase of glucosinolate formation. Or, it can undergo additional cycles ofacetyl-CoA condensation, isomerization, andoxidation-carboxylation, resulting in furtherelongation. Up to nine cycles are known tooccur in plants (49). Similar 2-oxo acid–basedchain-elongation sequences occur in leucinebiosynthesis and in the TCA cycle, as well aselsewhere in plant metabolism (89).The earliest evidence for the chainelongation pathway came from feeding studies with radiolabeled precursors beginning inthe 1960s (39, 97). More recent in vivo studies with stable isotopes (61, 62) confirmed theoutline and major intermediates of the pathway. Most critical was the observation thatacetate was readily incorporated into chainelongated amino acids with the additionalmethylene group derived exclusively from theC-2 position (acetate methyl group). The acetate carboxyl group is lost during chain elongation or during conversion into the coreglucosinolate. Additional support for thechain-elongation pathway was provided bythe detection of certain intermediates in thechain elongation of methionine (32) andphenylalanine (43) and by the isolation of thechain-elongated methionine homologs themselves (69). Furthermore, the activity of theaminotransferase producing the initial 2-oxoacid from methionine (33, 60) and the activityof the condensing enzyme of the first roundof methionine chain elongation (50) have beendemonstrated in cell-free extracts.The first information about the geneticbasis of chain elongation came from theidentification of a locus in Arabidopsis andBrassica napus that controls the chain lengthof methionine-derived glucosinolates (111).This locus was mapped in Arabidopsis using a cross between two ecotypes, Columbiaand Landsberg erecta, whose major glucosinolates are derived from dihomomethionineand homomethionine, respectively (29). The
Annu. Rev. Plant Biol. 2006.57:303-333. Downloaded from arjournals.annualreviews.orgby University of California - Davis on 10/15/08. For personal use only.ANRV274-PP57-12ARI27 March 200610:25Figure 2Amino acid chain-elongation cycle for glucosinolate biosynthesis. Illustrated is the first round ofelongation. The three principal steps are: (1) condensation with acetyl-CoA, (2) isomerization, and (3)oxidation-decarboxylation. The carbon atoms contributed by acetyl-CoA (retained with each round) areshown in red. The carbon atom from the original COOH function (lost with each round) is shown inblue.candidate genes were two adjacent sequenceswith high similarity to genes encoding isopropylmalate synthase, the enzyme catalyzingthe condensing reaction of chain elongation inleucine biosynthesis. Further fine-scale mapping identified one of the two genes, MAM1(Methylthioalkylmalate synthase 1), as responsible for the chain-elongation polymorphismin Columbia and Landsberg erecta (91). Thisfinding was confirmed by the isolation of missense mutants for this gene that had alteredglucosinolate chain-length profiles and theheterologous expression of MAM1 in E. coli,which gave an extract capable of condensingω-methylthio-2-oxoalkanoates with acetylCoA to give 2-(ω-methylthioalkyl)malates.The MAM1 gene product carried out the condensing reaction of only the first two methionine elongation cycles (152), suggesting thatthe second adjacent sequence (called MAM-Lfor “MAM-like”) might encode the proteinresponsible for the remaining activities. Indeed, a MAM-L knockout line was recently reported to lack long-chain methionine-derivedglucosinolates, but these were restored after transformation with a functional MAM-Lgene (51). A survey of Arabidopsis ecotypes revealed the presence of a third MAM-like gene,www.annualreviews.org Glucosinolates307
ANRV274-PP57-12ARI27 March 200610:25RHRCOOHRCYP79NNH 3NOHAmino OHNOHDesulfoglucosinolateS(Cys)C-S lyaseNAnnu. Rev. Plant Biol. 2006.57:303-333. Downloaded from arjournals.annualreviews.orgby University of California - Davis on 10/15/08. For personal use only.SHS -GTThiohydroximicacidOHS -alkylthiohydroximateSTSRGlcNOSO 3GlucosinolateFigure 3Biosynthesis of the glucosinolate core structure. CYP79 enzymescatalyzing the conversion of amino acids to aldoximes are the onlyside-chain-specific step in the pathway. The products from the CYP83sare too reactive to be isolated, but are proposed to be either aci-nitrocompounds or their dehydrated analogs, nitrile oxides. Thesulfur-donating enzyme is the only enzyme that remains to be identified,and is proposed to be a glutathione-S-transferase-like enzyme that usescysteine as substrate. Abbreviations: R, variable side chain; GST,glutathione-S-transferase; S-GT, S-glucosyltransferase;ST, sulfotransferase.designated MAM2 at the same locus (90).The majority of ecotypes examined possessedfunctional copies of either MAM1 or MAM2,but not both. A functional MAM1 sequencewas correlated with accumulation of glucosinolates having undergone two rounds of chainelongation, whereas a functional MAM2 sequence was correlated with accumulation ofglucosinolates having undergone only oneround of elongation. Our knowledge of aminoacid chain elongation has advanced rapidly inthe past five years, yet much more information about the genes and enzymes of this segment of glucosinolate biosynthesis is neces308Halkier·Gershenzonsary before we can fully understand how itis regulated and how substrates from primarymetabolism are channeled to the core pathwayof glucosinolate formation.BIOSYNTHESIS: CORESTRUCTUREThe biosynthesis of the core glucosinolatestructure involves intermediates common toall glucosinolates. Our knowledge on howamino acids are converted into the core glucosinolate structure has increased as researchhas advanced from traditional in vivo feeding studies and biochemical characterizationof the enzymatic activities in plant extractsto identification and characterization of thebiosynthetic genes encoding the enzymes.Here the presence of glucosinolates in themodel plant Arabidopsis has greatly facilitatedprogress. The intermediates in the pathwayfrom the amino acid to the core structure include N-hydroxy amino acids, aldoximes, acinitro or nitrile oxide compounds (both are tooreactive to be isolated), S-alkyl thiohydroximates, thiohydroximic acids, and desulfoglucosinolates (Figure 3). The genes responsible for all these steps, except the S-alkylation,have been identified since 2000.The Conversion of Amino Acids toAldoximesCytochromes P450 belonging to the CYP79family are responsible for catalyzing the conversion of amino acids to aldoximes (169).Most of the seven CYP79s in the glucosinolate pathway in Arabidopsis were identified using a functional genomics approach (66). Thiswas based on the similarity of the biosyntheticpathways of glucosinolates and cyanogenicglucosides, another group of amino acid–derived natural products with aldoximes as intermediates (78). As CYP79 homologs wereidentified in the Arabidopsis genome project,they were heterologously expressed and characterized with respect to substrate specificity(66).
Annu. Rev. Plant Biol. 2006.57:303-333. Downloaded from arjournals.annualreviews.orgby University of California - Davis on 10/15/08. For personal use only.ANRV274-PP57-12ARI27 March 200610:25The function of some CYP79 genes wasidentified using other approaches. A screenin yeast for cDNAs conferring resistanceto 5-fluoroindole (the precursor of a toxictryptophan derivative) led to isolation ofCYP79B2 (73), which together with the homolog CYP79B3 catalyze the conversion oftryptophan to indole-3-acetaldoxime (IAOx)(73, 117). A cyp79B2/cyp79B3 double knockout is completely devoid of indole glucosinolates (178), which shows that no other sourceof IAOx contributes significantly to biosynthesis of indole glucosinolate. Accordingly,the plasma membrane-bound peroxidasedependent conversion of tryptophan to IAOx(105, 106), and IAOx produced from theYUCCA pathway (177), are not involved inglucosinolate biosynthesis.In independent genetic approaches, twomutants, bushy (143) and supershoot (150), withsevere morphological alterations includingseveral hundred axillary shoots were shownto be knockout mutants of CYP79F1. Thesemutants completely lack short-chain aliphaticglucosinolates (143). Based on this finding,it was suggested that CYP79F1 metabolizesthe short-chain methionine derivatives (withone to four additional methylene groups),and that the homolog CYP79F2 that is 88%identical at the amino acid level metabolizesthe long-chain-elongated methionine derivatives (143). However, biochemical characterization of CYP79F1 and CYP79F2 showedthat CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- andlong-chain aliphatic glucosinolates, whereasCYP79F2 exclusively metabolizes long-chainpenta- and hexahomomethionines (34, 69).The substrate specificities of CYP79F1 andCYP79F2
1Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Denmark; email: bah@kvl.dk 2Department of Biochemistry, Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany; email: gershenzon@ice.mpg.de Annu. Rev. Plant Biol. 2006. 57:303–33 The Annual Review of
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