UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds

Abstract

We detected UDP-glucuronic acid:soyasapogenol glucuronosyltransferase (UGASGT) activity in the microsomal fraction from germinating soybean (Glycine max [L.] Merr.) seed. A microsomal fraction was isolated from germinating soybean seed and treated with various detergents to solubilize the enzyme. UGASGT activity was monitored throughout purification using UDP-[U-(14)C]glucuronic acid and soyasapogenol B as substrates. Purification of UGASGT was achieved by HiTrap Q, Superdex 200, and HiTrap Blue chromatography procedures. This resulted in >205-fold enrichment relative to the starting homogenate. UGASGT was found to require divalent cations for activity. Studies on the substrate specificity of UGASGT demonstrated that the specificity for the sugar residue transferred was very high, as activity was scarcely found when UDP-glucuronic acid was replaced by other UDP sugars: UDP-glucose and UDP-galactose. Soyasapogenols, which are the aglycons of soybean saponin, are usable acceptors, but glycyrrhetinic acid, sophoradiol, beta-amyrin, and flavonoids are not. These findings suggest that this UGASGT was a specific enzyme for UDP-glucuronic acid as a donor and soyasapogenols as acceptors, and that it was related to the biosynthesis of the sugar chain in soybean saponin. This study provides a basis for the molecular characterization of a key enzyme in saponin biosynthesis in soybean. The isolation of the gene may enable its use in the elucidation of the biosynthesis and physiological role of saponins in soybean.

UDP-glucuronic Acid:Anthocyanin Glucuronosyltransferase from Red Daisy (Bellis perennis) Flowers

“…It should be noted, however, that some PSPGs that are involved in xenobiotic metabolism also show low acceptor specificities (29,34), and the strict acceptor specificity of BpUGAT should be closely related to its specialized role in the biosynthesis of flower pigment. It should also be noted that the enzymatic properties of UDP-glucuronate:soyasapogenol UGAT of Glycine max (12) significantly differ from those of BpUGAT and UBGAT but are rather similar to those of vertebrate UGATs. For example, UDP-glucuronate:soyasapogenol UGAT appears to be a membrane-bound microsomal enzyme and requires Mg 2ϩ ion for catalytic activity (12).…”

“…It should also be noted that the enzymatic properties of UDP-glucuronate:soyasapogenol UGAT of Glycine max (12) significantly differ from those of BpUGAT and UBGAT but are rather similar to those of vertebrate UGATs. For example, UDP-glucuronate:soyasapogenol UGAT appears to be a membrane-bound microsomal enzyme and requires Mg 2ϩ ion for catalytic activity (12). This might be related to a difference in their phylogenetic positions.…”

“…UGATs are also involved in plant secondary metabolism of biomedical and agricultural relevance, such as the biosynthesis of baicalin (5,6-dihydroxy-7-O-glucuronosylflavone), a major flavonoid of the dried roots of Scutellaria baicalensis, that shows antiallergic, anti-HIV, and antitumor activities (10). Some of these UGATs (11,12), including UDP-glucuronate:baicalein 7-UGAT (UBGAT) of S. baicalensis (10), have been purified and characterized. However, the genetic aspects of these UGATs have yet to be studied, although the primary structure of UBGAT has recently been submitted to data bases (GenBank TM /DDBJ/EBI accession number BAC98300).…”

Local Differentiation of Sugar Donor Specificity of Flavonoid Glycosyltransferase in Lamiales

“…The positional plasticity of the crucial Arg residue not only highlights a convergent evolution of sugar donor specificity of UGT, but it also suggests that the GAT gene cannot be assigned based solely on the position of the Arg-25 and Arg-350. Thus, it is possible that another Arg residue may be present in unidentified GATs and that it is catalyzing the glucuronosylation of phytochemicals beneficial for human life (for example, the soybean's soyasapogenol, green tea's theasaponin, licorice's glycyrrhizin, and St. John's wort's miquelianin) (Butterweck et al, 2000;Kurosawa et al, 2002;Yoshikawa et al, 2007;Seki et al, 2008). A homology modeling strategy will help identify the crucial residue based on the biochemical basis of GAT.…”

Legume Natural Products: Understanding and Manipulating Complex Pathways for Human and Animal Health

“…The aglycones are subsequently converted to the saponins by the action of a series of glycosyltransferases (GTs). To date, only a single GT involved in saponin biosynthesis in soybean has been characterized biochemically (Kurosawa et al, 2002). This pathway is a prime candidate for functional genomics approaches (see below).…”