Purification and characterization of UDP-glucose: anthocyanin 3',5'-O-glucosyltransferase from Clitoria ternatea.
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.
Division of Cell Engineering, Aomori Green BioCentre, 221-10 Nogi-Yamaguchi, Aomori, Aomori 030-0142, Japan.
Koichiro
K
Kogawa
Naoki
N
Kato
Kohei
K
Kazuma
Naonobu
N
Noda
Masahiko
M
Suzuki
D000872:Anthocyanins / Q000737:chemistry / Q000378:metabolism*; D031292:Clitoria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
D000872:Anthocyanins; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose