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Abstract

S-Alk(en)yl-L-cysteine sulfoxides are pharmaceutically important secondary metabolites produced by plants that belong to the genus Allium. Biosynthesis of S-alk(en)yl-L-cysteine sulfoxides is initiated by S-alk(en)ylation of glutathione, which is followed by the removal of glycyl and γ-glutamyl groups and S-oxygenation. However, most of the enzymes involved in the biosynthesis of S-alk(en)yl-L-cysteine sulfoxides in Allium plants have not been identified. In this study, we identified three genes, AsGGT1, AsGGT2, and AsGGT3, from garlic (Allium sativum) that encode γ-glutamyl transpeptidases (GGTs) catalyzing the removal of the γ-glutamyl moiety from a putative biosynthetic intermediate of S-allyl-L-cysteine sulfoxide (alliin). The recombinant proteins of AsGGT1, AsGGT2, and AsGGT3 exhibited considerable deglutamylation activity toward a putative alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine, whereas these proteins showed very low deglutamylation activity toward another possible alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine sulfoxide. The deglutamylation activities of AsGGT1, AsGGT2, and AsGGT3 toward γ-glutamyl-S-allyl-L-cysteine were elevated in the presence of the dipeptide glycylglycine as a γ-glutamyl acceptor substrate, although these proteins can act as hydrolases in the absence of a proper acceptor substrate, except water. The apparent Km values of AsGGT1, AsGGT2, and AsGGT3 for γ-glutamyl-S-allyl-L-cysteine were 86 μM, 1.1 mM, and 9.4 mM, respectively. Subcellular distribution of GFP-fusion proteins transiently expressed in onion cells suggested that AsGGT2 localizes in the vacuole, whereas AsGGT1 and AsGGT3 possess no apparent transit peptide for localization to intracellular organelles. The different kinetic properties and subcellular localizations of AsGGT1, AsGGT2, and AsGGT3 suggest that these three GGTs may contribute differently to the biosynthesis of alliin in garlic.

ASSAYS OF GGT ENZYME ACTIVITIES

For the analysis of deglutamylation activities toward γ-glutamyl-S-allyl-L-cysteine and γ-glutamyl-S-allyl-L-cysteine sulfoxide, the enzyme assay reaction mixture consisted of 0.67 μg μl-1 yeast crude protein, 50 mM Tris-HCl (pH 8.0), 10 mM glycylglycine, and 1 mM γ-glutamyl-S-allyl-L-cysteine or γ-glutamyl-S-allyl-L-cysteine sulfoxide was incubated for 6 h at 37°C. For the determination of the enzyme activity in a pH range of 6.0–7.0, the reaction mixture containing 50 mM 2-(N-morpholino)ethanesulfonic acid buffer, instead of 50 mM Tris-HCl buffer, was used. For the determination of the enzyme activity in a pH range of 7.0–9.0, 50 mM Tris-HCl buffer at pH values from 7.0 to 9.0, instead of 50 mM Tris-HCl (pH 8.0), was used. For the analysis of the effects of glycylglycine as a γ-glutamyl acceptor, deglutamylation activity was determined in the reaction mixture with or without 10 mM glycylglycine. The reaction was initiated by the addition of yeast crude proteins. After incubation at 37°C, proteins in the reaction mixture were removed using a centrifugal ultrafiltration device (molecular weight cut-off, 10 kD; Kurabo, Osaka, Japan). S-Allyl-L-cysteine and alliin in the ultrafiltrated solution were quantified using high-performance liquid chromatography (HPLC). For the kinetic analysis, assays were carried out with γ-glutamyl-S-allyl-L-cysteine concentrations ranging from 12.5 to 1000 μM for AsGGT1, 0.5–8 mM for AsGGT2, and 1–25 mM for AsGGT3. Km values were calculated from triplicate date sets according to the Michaelis-Menten equation.