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

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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)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-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 K m value for 1 (3.89 × 10−5 M), which is lower than that for 2 (1.38 × 10−4 M), renders the k cat/K m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K m value for UDP-glucose (6.18 × 10−3 M) with saturated 2 is larger than that for UDP-glucose (1.49 × 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.

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Correspondence to Kohei Kazuma.

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K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.

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Kogawa, K., Kato, N., Kazuma, K. et al. Purification and characterization of UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase from Clitoria ternatea . Planta 226, 1501–1509 (2007).

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