Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties (original) (raw)
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Biochimica et biophysica acta, 1972
Myrosinases (EC 3.2.3.1) are glucosinolate (thioglucoside) hydrolases primarily occurring in plants of the Cruciferae family. I. The separation of three myrosinase isoenzymes in Sin@is alba seed was achieved by DEAE-cellulose chromatography on Whatman DE-52. 2. The main myrosinase component was completely purified by DEAE-cellulose chromatography followed by gel chromatography on Sephadex G-2oo and isoelectrie focusing in an LKB-column. 3. The isolated myrosinase was found to be a glycoprotein with a molecular weight of 151 ooo, consisting of two identical polypeptide subunits with a molecular weight of 62 ooo each and a carbohydrate part. The electrophoretic mobility was determined to 7.0. IO-4 cm2/s per V and the isoelectric point was found to be pH 5.08.
Myrosinase Activity in Differentiated and Undifferentiated Plants of Brassicaceae
Zeitschrift für Pflanzenphysiologie, 1980
The occurrence and distribution of myrosinases in different plant organs in several species of Brassicaceae (Brassica chinensis, B. napus L., B. oleracea L., lberis amara L., lberis amara var. coronaria, Lepidium sativum L., Raphanus sativus L. and Sinapis alba L.) was studied. Myrosinase solutions were prepared from roots, stems, leaves, callus cultures and protoplasts and after dialysis the enzymatic activity was detected after hydrolytic cleavage of sinigrin. Specific activity was calculated in relation to protein, and it was found to vary within the range of 0.490 to 0.010 ,umol. min-I. mg-1 in 1 to 2 day old seedlings. The activity was found to decrease throughout the development of the plant but even at the flowering stage myrosinase activity could be detected. In callus cultures originally isolated from stem segments of wild candytuft and chinese cabbage myrosinase activity was demonstrated 48 and 64 weeks, respectively, after the start of the cultivation period. Protoplasts from the abovementioned species were also isolated from leaf tissue and their morphological shape examined by light-and scanning electron microscopy. Although myrosinase activity was not detectable in protoplasts of cabbage, cress and rape, the myrosinase extracts from protoplasts of the other species showed higher specific activity than the in tact leaf cells. The distribution of myrosinases has been interpreted and correlated to the development stage of the plant tissue.
Phytochemistry, 2014
The pungent taste of horseradish is caused by isothiocyanates which are released from glucosinolates by myrosinases. These enzymes are encoded by genes belonging to one of two subfamilies, termed MYR I and MYR II, respectively. A MYR II-type myrosinase gene was identified for the first time in horseradish. The gene termed ArMY2 was only expressed in young roots. A full-length cDNA encoding a myrosinase termed ArMy2 was isolated and heterologously expressed in Pichia pastoris. The recombinant His-tagged enzyme was characterized biochemically. Substrate affinity was 5 times higher towards gluconasturtiin than towards sinigrin. Gluconasturtiin was found to be the most abundant glucosinolate in young horseradish roots while sinigrin dominated in storage roots and leaves. This indicates that a specialized glucosinolate-myrosinase defense system might be active in young roots.
THE GLUCOSINOLATES-MYROSINASE SYSTEM: FROM CHEMISTRY, BIOLOGY TO ECOLOGY
Glucosinolates, a well-defined, sulfur-rich class of plant secondary products mainly confined to various crops of the family Brassicaceae, are of prime importance in agriculture and plant biotechnology since the discovery of their role in plant defense against insect herbivory. Till date more than 120 different types of the compound have been reported. The enzyme myrosinase (thioglucosidases), which is stored in specialized plant cells, converts glucosinolates to the several toxic products (e.g., isothiocyanates, thiocyanates, and nitriles). The hydrolysis products have many different biological activities for plants, e.g., as defense compounds as well as attractants. In case of human, they may play several roles as biopesticides, flavor compounds and cancer-preventing agents. In the present article, we try to discuss broadly the biochemistry and the roles of the compounds, their break-down products in the insect-plant relationships and multitrophic interactions. Major focus has been laid on Brassicaceous crop plants, where they are most abundantly found.
Journal of agricultural and food chemistry, 2014
Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.
Plant molecular biology, 2004
A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific express...
Industrial Crops and Products, 2013
Myrosinase (EC 3.2.3.1) found in Brassicaceae plants, is the enzyme responsible for hydrolysis of glu-cosinolates. As a result a variety of biologically active metabolites are liberated, whose importance in crop protection and especially in cancer chemoprevention is rapidly gaining recognition. The growing practical application of glucosinolate degradation products requires that sensitive and reliable methods of myrosinase activity determination in different types of plant samples are established. With the use of commercial myrosinase prep, we systematically optimised conditions of measurement of this enzyme activity by spectrophotometric and pH-stat methods. The parameters evaluated included: sample preparation, choice of substrate, its concentration, reaction temperature and detection wavelength. Two substrates with different spectral properties were chosen: sinigrin (SIN) and glucotropaeolin (GTL). For both substrates, the best reliability was achieved at reaction temperature of 37 • C and substrate concentration of 0.2 mM and 5mM for spectrophotometric and pH-stat methods, respectively. GTL exhibiting higher absorption at the recommended detection wavelength of 230 nm ensured greater sensitivity of spectrophotometric determination of myrosinase activity in the case of transparent plant samples. GTL seemed to increase also the sensitivity of pH-stat method, however, in this case homogenisation of plant samples turned out to be most important. The optimised conditions were then verified for a range of plant samples. Based on these results, the optimised protocols of myrosinase activity determination for both methods are proposed.
Insect Biochemistry and Molecular Biology, 2005
The aphid Brevicoryne brassicae is a specialist feeding on Brassicaceae plants. The insect has an intricate defence system involving a b-D-thioglucosidase (myrosinase) that hydrolyses glucosinolates sequestered from the host plant into volatile isothiocyanates. These isothiocyanates act synergistically with the pheromone E-b-farnesene to form an alarm system when the aphid is predated. In order to investigate the enzymatic characteristics of the aphid myrosinase and its three-dimensional structure, milligram amounts of pure recombinant aphid myrosinase were obtained from Echerichia coli. The recombinant enzyme had similar physiochemical properties to the native enzyme. The global structure is very similar to Sinapis alba myrosinase and plant b-O-glucosidases. Aphid myrosinase has two catalytic glutamic acid residues positioned as in plant b-O-glucosidases, and it is not obvious why this unusual enzyme hydrolyses glucosinolates, the common substrates of plant myrosinases which are normally not hydrolyzed by plant b-Oglucosidases. The only residue specific for aphid myrosinase in proximity of the glycosidic linkage is Tyr180 which may have a catalytic role. The aglycon binding site differs strongly from plant myrosinase, whereas due to the presence of Trp424 in the glucose binding site, this part of the active site is more similar to plant b-O-glucosidases, as plant myrosinases carry a phenylalanine residue at this position. r
Properties of β-thioglucoside hydrolases (TGG1 and TGG2) from leaves of< i> Arabidopsis thaliana
Plant Science, 2012
Myrosinases (-thioglucoside glucohydrolase, TGG; EC 3.2.1.147) catalyze the hydrolysis of glucosinolates, a structurally distinct group of nitrogen-and sulfur-containing secondary metabolites, to give a chemically unstable intermediate, glucose and sulfate. This catalysis initiates a chemical defense in crucifer plants as a response to the tissue-damaging activities of herbivores and pathogens. To characterize the individual and collective biochemical properties of the myrosinase enzymes found in the aerial tissues of Arabidopsis thaliana, we purified TGG1 and TGG2, which share 73% amino acid identity, individually from T-DNA insertion lines of Arabidopsis using lectin affinity and anion exchange chromatography. Electrophoresis under denaturing conditions and the mobility of undenatured TGG1 and TGG2 protein on gel filtration chromatography indicated that the native proteins exist as dimers of 150 and 126 kDa, respectively.
2001
also present in seedling cotyledons. However, from this tissue, Extraction of Sinapis alba seeds under native conditions soluthey could be extracted with non-denaturing buffers. In addi-bilized 3 myrosinase isoforms, pool I, II and III, which could tion, cotyledons contained a 65-kDa MB myrosinase not be separated by ion exchange chromatography. Sequencing of found in seeds. In contrast, seedling cotyledons contained only numerous peptides of the I and III isoforms showed that they minute amounts of pool I and no pool III MA myrosinases, belonged to the Myrosinase A (MA) family of myrosinases emphasizing the tissue-specific expression of the corresponding and that they were encoded by different genes. Western blot analysis of S. alba seed proteins, extracted with a sodium gene families. Sequence analysis of myrosinase cDNAs generated cDNA by reversed transcription-polymerase chain reac-dodecyl sulphate-containing buffer, using an anti-myrosinase tion using degenerate primers with mRNA isolated from seeds, monoclonal antibody, showed the presence of two additional cotyledons and leaves confirmed the result that the MA myrosinase isoforms with approximate molecular sizes of 62 and 59 kDa. These myrosinases, which only could be solubi-isoforms were expressed only in seed tissue, while MB myrolized from seeds by inclusion of denaturing agents in the sinases were found in all tissues investigated. Furthermore, extraction buffer, were by sequence analysis identified as MB seed and leaf contained unique MB myrosinase transcripts, myrosinases. These isoenzymes or very similar forms were suggesting organ-specific expression of individual MB genes.