Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties.
Myrosinases (EC 3.2.1.147) are beta-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1-TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS-PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation-inhibition responses towards ascorbic acid with maximal activity around 0.7-1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-beta-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.
Andersson
Derek
D
Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Box 7080, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden.
Chakrabarty
Romit
R
Bejai
Sarosh
S
Zhang
Jiaming
J
Rask
Lars
L
Meijer
Johan
J
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism; D002384:Catalysis; D018076:DNA, Complementary; D017343:Genes, Plant; D005961:Glucosinolates / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012492:Salts; D013696:Temperature; D001617:beta-Glucosidase / Q000378:metabolism
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D005961:Glucosinolates; D007527:Isoenzymes; D011994:Recombinant Proteins; D012492:Salts; C010330:sinigrin; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D001205:Ascorbic Acid