Purification and properties of 3′-phosphoadenosine-5′-phosphosulphate: Desulphoglucosinolate sulphotransferase from Brassica juncea cell cultures

Jain, Jinesh C.; GrootWassink, J.W.D.; Kolenovsky, Allan; Underhill, E. W.

doi:10.1016/0031-9422(90)80094-w

Cited by 27 publications

(19 citation statements)

References 12 publications

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“…2) were made in the expression vector pSP19g10L (Ref. 27; kindly provided by Dr. Henry Barnes, Synthetic Genetics/Immune Complex Inc., San Diego, CA): 79A2 ("native"), 17A (1)(2)(3)(4)(5)(6)(7)(8) 79A2 ("modified"), 17A (1)(2)(3)(4)(5)(6)(7)(8) 79A2⌬ (1)(2)(3)(4)(5)(6)(7)(8) ("truncated-modified"), and 17A (1)(2)(3)(4)(5)(6)(7)(8) 79A1 79A2⌬(1-40) ("chimeric") (79A2: CYP79A2; 17A (1)(2)(3)(4)(5)(6)(7)(8): modified N terminus of CYP17A, MALLLAVF (Ref. 28); 79A1 : amino acids 25-74 of CYP79A1 (Ref.…”

Section: Methodsmentioning

confidence: 99%

“…6). The enzymes catalyzing the last two steps in the pathway, UDPG: thiohydroximate glucosyltransferase (EC 2.4.1.-) and 3Ј-phosphoadenosine 5Ј-phosphosulfate:desulfoglucosinolate sulfotransferase (EC 2.8.2.-), have been purified and shown to be nonspecific with respect to the nature of the side chain (7)(8)(9)(10). The sulfur-donating enzyme has not been characterized, but feeding experiments suggest that cysteine is the sulfur donor (11).…”

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confidence: 99%

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Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate

Wittstock¹,

Halkier²

2000

Journal of Biological Chemistry

234

161

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Glucosinolates are natural plant products gaining increasing interest as cancer-preventing agents and crop protectants. Similar to cyanogenic glucosides, glucosinolates are derived from amino acids and have aldoximes as intermediates. We report cloning and characterization of cytochrome P450 CYP79A2 involved in aldoxime formation in the glucosinolate-producing Arabidopsis thaliana L. The CYP79A2 cDNA was cloned by polymerase chain reaction, and CYP79A2 was functionally expressed in Escherichia coli. Characterization of the recombinant protein shows that CYP79A2 is an Nhydroxylase converting L-phenylalanine into phenylacetaldoxime, the precursor of benzylglucosinolate. Transgenic A. thaliana constitutively expressing CYP79A2 accumulate high levels of benzylglucosinolate. CYP79A2 expressed in E. coli has a K m of 6.7 mol liter ؊1 for L-phenylalanine. Neither L-tyrosine, L-tryptophan, Lmethionine, nor DL-homophenylalanine are metabolized by CYP79A2, indicating that the enzyme has a narrow substrate specificity. CYP79A2 is the first enzyme shown to catalyze the conversion of an amino acid to the aldoxime in the biosynthesis of glucosinolates. Our data provide the first conclusive evidence that evolutionarily conserved cytochromes P450 catalyze this step common for the biosynthetic pathways of glucosinolates and cyanogenic glucosides. This strongly indicates that the biosynthesis of glucosinolates has evolved based on a cyanogenic predisposition.Glucosinolates are amino acid-derived, secondary plant products containing a sulfate and a thioglucose moiety. Glucosinolates are found throughout the order Capparales, which includes agriculturally important crops of the Brassicaceae family such as oilseed rape and Brassica forages and vegetables, and the model plant Arabidopsis thaliana L. Upon tissue damage, glucosinolates are rapidly hydrolyzed to biologically active degradation products by the thioglucosidase myrosinase (EC 3.2.3

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate

Wittstock¹,

Halkier²

2000

Journal of Biological Chemistry

234

161

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“…The last two steps in the pathway are catalyzed by two soluble enzymes, the UDPG-thiohydroximate glucosyltransferase (EC 2.4.1.-), which glucosylates the thiohydroximate, and the 3'-phosphoadenosine 5'-phosphosulfate:desulfoglucosinolate sulfotransferase (EC 2.8.2.-), which converts the desulfoglucosinolate into the glucosinolate. These two enzymes have been purified and shown to be nonspecific with respect to the nature of the side chain (Glendening and Poulton, 1988;Jain et al, 1990;Reed et al, 1993;Guo and Poulton, 1994). The sulfur-donating enzyme has not been characterized, but feeding experiments suggest that Cys is the sulfur donor (Matsuo, 1968).…”

Section: ' This Work Was Partially Supported By the Center For Plantmentioning

confidence: 99%

Isolation of a Microsomal Enzyme System Involved in Glucosinolate Biosynthesis from Seedlings of Tropaeolum majus L

Du¹,

Halkier²

1996

Plant Physiology

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An in vitro system that converts phenylalanine to phenylacetaldoxime in the biosynthesis of the glucosinolate glucotropaeolin has been established in seedlings of Tropaeolum majus L. exposed to the combined treatment of jasmonic acid, ethanol, and light. The treatment resulted in a 9-fold induction, compared with untreated, dark-grown seedlings, of de novo biosynthesis measured as incorporation of radioactively labeled phenylalanine into glucotropaeolin. Formation of the inhibitory degradation product benzylisothiocyanate during tissue homogenization was prevented by inactivation of the thioglucosidase myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. This allowed the isolation of a biosynthetically active microsomal preparation from the induced T. majus plant material. The enzyme, which catalyzes the conversion of phenylalanine to the corresponding oxime, was sensitive to cytochrome P450 inhibitors, indicating the involvement of a cytochrome P450 in the biosynthetic pathway. It has previously been shown that the oxime-producing enzyme in the biosynthesis of p-hydroxybenzylglucosinolate in Sinapis alba L. is dependent on cytochrome P450, whereas the oxime-producing enzymes in Brassica species have been suggested to be flavin monooxygenases or peroxidase-type enzymes. The result with T. majus provides additional experimental documentation for a similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glucosides.

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“…The final step in the biosynthesis of the GS core structure is catalyzed by a desulfoglucosinolate:PAPS sulfotransferase (dsGS-ST), transferring the sulfate moiety from PAPS to the desulfoglucosinolate (dsGS). The enzymatic activity of dsGS-STs has been analyzed in partially purified protein fractions from Brassica juncea and cress (Lepidium sativum) (22,23). The enzymes have similar biochemical characteristics with respect to native molecular mass, isoelectric point, pH, and temperature optima as well as inhibition by various sulfhydryl group reagents.…”

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confidence: 99%

Desulfoglucosinolate Sulfotransferases from Arabidopsis thaliana Catalyze the Final Step in the Biosynthesis of the Glucosinolate Core Structure

Piotrowski

Schemenewitz

Lopukhina

et al. 2004

Journal of Biological Chemistry

181

155

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The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His 6 -tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan-and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.Compared with the animal cell, very little is known regarding the structural and regulatory roles of the sulfate group in plants. The transfer of the active sulfate group from 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) 1 to acceptor molecules is catalyzed by sulfotransferases. Members of the superfamily of sulfotransferases are known in prokaryotes as well as eukaryotes; however, the study of enzymes that catalyze the sulfation reaction in plants considerably lags behind that in animal systems. Cytosolic sulfotransferases from plants have been characterized in some detail (Ref. 1 and references therein), and some cDNAs have been identified. These fall into three subgroups: the flavonol sulfotransferases described for Flaveria species (1), the steroid sulfotransferases identified in Brassica napus (2, 3), and a hydroxyjasmonic acid-specific sulfotransferase from Arabidopsis thaliana (4). An additional sulfotransferase (RaR047, At2g03760) has been cloned from A. thaliana, and its mRNA level was found to be up-regulated by pathogens and salicylic acid; however, its physiological substrate is still unknown (5).In plants, sulfate groups occur in a number of secondary metabolites, notably the sulfoflavonoids (6) and the glucosinolates (7). Glucosinolates (GSs) are secondary compounds found in at least 16 different plant families, 15 of which belong to the order Cappa...

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Purification and properties of 3′-phosphoadenosine-5′-phosphosulphate: Desulphoglucosinolate sulphotransferase from Brassica juncea cell cultures

Cited by 27 publications

References 12 publications

Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate

Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate

Isolation of a Microsomal Enzyme System Involved in Glucosinolate Biosynthesis from Seedlings of Tropaeolum majus L

Desulfoglucosinolate Sulfotransferases from Arabidopsis thaliana Catalyze the Final Step in the Biosynthesis of the Glucosinolate Core Structure

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