Partial purification and characterization of a 3′-phosphoadenosine 5′-phosphosulfate: Desulfoglucosinolate sulfotransferase from cress (Lepidium sativum)

Abstract

A 3′-phosphoadenosine 5′-phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (Km′ 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (Km′ 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (Km′ 670 micromolar), and desulfoallylglucosinolate (KM′ 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl2, MnCl2 and reducing agents and inhibited by ZnCl2, PbNO3′ NiCl2′ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′-dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.