Purification and characterization of UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase from Clitoria ternatea

Abstract

A UDP-glucose: anthocyanin 3',5'-O-glucosyltransferase (UA3'5'GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6''-O-malonyl)-beta-glucoside (1) is first converted to delphinidin 3-O-(6''-O-malonyl)-beta-glucoside-3'-O-beta-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6''-O-malonyl)-beta-glucoside-3',5'-di-O-beta-glucoside. UA3'5'GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin's 3'- or 5'-OH groups. Its kinetic properties showed comparable k (cat) values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent Km value for 1 (3.89 x 10(-5) M), which is lower than that for 2 (1.38 x 10(-4) M), renders the k(cat)/Km value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent Km value for UDP-glucose (6.18 x 10(-3) M) with saturated 2 is larger than that for UDP-glucose (1.49 x 10(-3) M) with saturated 1, the k(cat) values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3'5'GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1' single bond axis so that the 5'-OH group of 2 can point toward the catalytic center.

Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.

“…The addition of CuSO 4 , FeSO 4 , or ZnSO 4 to 0.5 mM concentration led to the complete loss of UGT90A7 and UGT95A1 activities, while UGT72B11 activity was only reduced to 12, 13, and 89%, respectively. Many Xavonoid glucosyltransferases are inhibited by Zn 2+ , while some anthocyanidin-O-glucosyltransferases are known to keep up moderate activity in the presence of even 1 mM Zn 2+ (Kamsteeg et al 1978;Yabuya et al 2002;Fukuchi-Mizutani et al 2003;Kogawa et al 2007). CoSO 4 at 0.5 mM almost completely inhibited UGT95A1, whereas UGT72B11 and UGT90A7 exhibited commensurable activities (38 and 45%) at the same concentration.…”

Crystal structure of UDP-glucose:anthocyanidin 3-O-glucosyltransferase fromClitoria ternatea

“…Studies on ternatin biosynthesis in C. ternatea revealed that delphinidin is not directly glucosylated at the 3 0 -or 5 0 -hydroxyl group, but that glucosylation of delphinidin occurs only when it has a 6 00 -Omalonyl--glucoside at the 3-position. Thus, glucosylation of delphinidin at the 3-hydroxyl group was proposed to be the first key step of ternatin biosynthesis (Kogawa et al, 2007).…”

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

“…Biochemical studies seem to support regiospecificity as a key parameter for UGT substrate specificity. At the same time it is also evident that the presence of a specific structural domain will not by itself assure activity, but should be considered in conjunction with the overall structure of the aglycone (Richman et al, 2005;He et al, 2006;Kogawa et al, 2007;Modolo et al, 2007). Several studies of biochemically described UGTs show some conservation of regiospecificity within phylogenetic groups (Vogt and Jones, 2000;Lim et al, 2003a;Morita et al, 2005;Yonekura-Sakakibara et al, 2007;Caputi et al, 2008).…”