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Abstract

Sinapine is a bitter phenolic choline ester that is ubiquitous in crucifer seeds. It occupies about 1-4% of the dry matter of rapeseed. Elimination of sinapine from the seeds would improve the flavor, palatability and nutritional properties of rapeseed meal for its utilization as supplemental protein source for animal feed. UDP-glucose:sinapic acid glucosyltransferase (SGT; EC 2.4.1.120) is a key enzyme involved in biosynthesis of both sinapine in seed and sinapoylmalate in vegetative tissues of many members of Brassicaceae. Both esters are strongly UV-absorbing but their physiological functions in plants are unknown. By a series of column chromatography techniques, SGT has been purified from 60-h-old seedlings of Brassica napus and characterized. SGT is a monomeric polypeptide with a molecular weight of 42 kDa and a pi of pH 5. The subcellular location of SGT appears to be the cytosol. SGT activity is not inducible by heat shock or UV radiation stresses. Its general characteristics are similar to those of SGT from Raphanus sativus, as well as a number of other UDP-glucose-dependent glucosyltransferases. The K[sub m (UDP-glucose)] was 0.24 mM and K[sub m (sinapic acid)] was 0.16 mM. SGT also catalyzes the reverse reaction in vitro, using UDP and sinapoylglucose to form UDP-glucose. No cofactors are required for SGT activity, but reducing reagents and glycerol are required to stabilize the enzyme. The enzyme is strongly inhibited by phydroxyl-mercuribenzoic acid, UDP, TDP, Zn⁺⁺, Cu⁺⁺ and Hg⁺⁺. Kinetic properties and substrate affinity data suggest that the catalytic mechanism of SGT is best described by a "random bi-bi" model. An analysis of developmental profiles showed that SGT was expressed in all growth stages of B. napus plants, but was most active during the early germination and seed development stages, particularly in cotyledons, juvenile leaves and young shoots. Partial amino acid sequence data were obtained from tryptic digests of the putative SGT protein. These sequences showed a very high degree of similarity to the HSP/HSC70 protein family.