Vogt T, Zimmermann E, Grimm R, Meyer M, Strack D
Institut für Pflanzcnbiochemie (IPB), Abteilung Sekundärstoffwechsel, Halle, Saale, Germany.
Planta. 1997;203(3):349-61. doi: 10.1007/s004250050201.
Uridine 5'-diphosphoglucose:betanidin 5-O- and 6-O-glucosyltransferases (5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific formation of betanin (betanidin 5-O-beta-glucoside) and gomphrenin I (betanidin 6-O-beta-glucoside), respectively. Both enzymes were purified to near homogeneity from cell-suspension cultures of Dorotheanthus bellidiformis, the 5-GT by classical chromatographic techniques and the 6-GT by affinity dye-ligand chromatography using UDP-glucose as eluent. Data obtained with highly purified enzymes indicate that 5-GT and 6-GT catalyze the indiscriminate transfer of glucose from UDP-glucose to hydroxyl groups of betanidin, flavonols, anthocyanidins and flavones, but discriminate between individual hydroxyl groups of the respective acceptor compounds. The 5-GT catalyzes the transfer of glucose to the C-4' hydroxyl group of quercetin as its best substrate, and the 6-GT to the C-3 hydroxyl group of cyanidin as its best substrate. Both enzymes also catalyze the formation of the respective 7-O-glucosides, but to a minor extent. Although the enzymes were not isolated to homogeneity, chromatographic, electrophoretic and kinetic properties proved that the respective enzyme activities were based on the presence of single enzymes, i.e. 5-GT and 6-GT. The N terminus of the 6-GT revealed high sequence identity to a proposed UDP-glucose: flavonol 3-O-glucosyltransferase (UF3GT) of Manihot esculenta. In addition to the 5-GT and 6-GT, we isolated a UF3GT from D. bellidiformis cell cultures that preferentially accepted myricetin and quercetin, but was inactive with betanidin. The same result was obtained with a UF3GT from Antirrhinum majus and a flavonol 4'-O-glucosyltransferase from Allium cepa. Based on these results, the main question to be addressed reads: Are the characteristics of the 5-GT and 6-GT indicative of their phylogenetic relationship with flavonoid glucosyltransferases?
尿苷5'-二磷酸葡萄糖：甜菜色素苷5-O-和6-O-葡萄糖基转移酶（5-GT和6-GT；EC 2.4.1）分别催化甜菜红素（甜菜色素苷5-O-β-葡萄糖苷）和千日红素I（甜菜色素苷6-O-β-葡萄糖苷）的区域特异性形成。两种酶均从龙须海棠的细胞悬浮培养物中纯化至接近均一，5-GT采用经典色谱技术纯化，6-GT采用以UDP-葡萄糖为洗脱剂的亲和染料配体色谱法纯化。用高度纯化的酶获得的数据表明，5-GT和6-GT催化UDP-葡萄糖中的葡萄糖无差别地转移至甜菜色素苷、黄酮醇、花青素和黄酮的羟基上，但对各自受体化合物的单个羟基有选择性。5-GT催化葡萄糖转移至槲皮素的C-4'羟基上，作为其最佳底物，而6-GT催化葡萄糖转移至矢车菊素的C-3羟基上，作为其最佳底物。两种酶也催化各自7-O-葡萄糖苷的形成，但程度较小。尽管这些酶未纯化至均一，但色谱、电泳和动力学性质证明各自的酶活性基于单一酶的存在，即5-GT和6-GT。6-GT的N末端与木薯中一种假定的UDP-葡萄糖：黄酮醇3-O-葡萄糖基转移酶（UF3GT）具有高度序列同一性。除了5-GT和6-GT，我们还从龙须海棠细胞培养物中分离出一种UF3GT，它优先接受杨梅黄酮和槲皮素，但对甜菜色素苷无活性。金鱼草的一种UF3GT和洋葱的一种黄酮醇4'-O-葡萄糖基转移酶也得到了相同结果。基于这些结果，要解决的主要问题是：5-GT和6-GT的特性是否表明它们与类黄酮葡萄糖基转移酶的系统发育关系？
