Purification and properties of UDP-glucose:thiohydroximate glucosyltransferase from Brassica napus L. seedlings

Arch Biochem Biophys. 1993 Sep;305(2):526-32. doi: 10.1006/abbi.1993.1456.

Abstract

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.

MeSH terms

  • Brassica / enzymology*
  • Glucosinolates / metabolism
  • Glucosyltransferases / isolation & purification*
  • Glucosyltransferases / metabolism
  • Hydrogen-Ion Concentration
  • Isoelectric Point
  • Metals
  • Molecular Weight
  • Plant Proteins / isolation & purification
  • Substrate Specificity

Substances

  • Glucosinolates
  • Metals
  • Plant Proteins
  • Glucosyltransferases
  • UPD-glucose-thiohydroximate glucosyltransferase