Purification and properties of UDP-glucose:thiohydroximate glucosyltransferase from Brassica napus L. seedlings.
A uridinediphosphateglucose:thiohydroximate glucosyltransferase (EC 2.4.1.-) has been purified 3700-fold from Brassica napus L. seedlings. The enzyme catalyzes the formation of desulfoglucosinolates by transfer of glucose from UDP-glucose to thiohydroximates and is believed to be the second to last step involved in glucosinolate biosynthesis. The enzyme was purified to near homogeneity, exhibiting a single band by non-denaturing polyacrylamide gel electrophoresis (PAGE) and on sodium dodecyl sulfate-PAGE (M(r) 46,000) but showed multiple isoforms between pH 4.6 and 4.3 when resolved by IEF. The enzyme is stable at temperatures up to 30 degrees C for at least 1 h and shows maximum activity rates at pH 6.0 and has no absolute requirements for cations. The Km values for UDP-glucose and phenylacetothiohydroximate were calculated to be 0.46 and 0.05 mM, respectively. This enzyme possesses a high degree of specificity for the thiohydroximic functional group but little specificity for the associated side-chain groups. Similar enzyme activity has been detected in all other members of the Brassicaceae family tested and is believed to be a common thiohydroximate glucosylating enzyme present in these and other glucosinolate producing plants.
National Research Council, Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada.
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Reed
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Davin
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Jain
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Deluca
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Nelson
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Underhill
D001937:Brassica / Q000201:enzymology*; D005961:Glucosinolates / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008670:Metals; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity
D005961:Glucosinolates; D008670:Metals; D010940:Plant Proteins; D005964:Glucosyltransferases; C059880:UPD-glucose-thiohydroximate glucosyltransferase