Mutational analysis of the Medicago glycosyltransferase UGT71G1 reveals residues that control regioselectivity for (Iso) flavonoid glycosylation
Mutational analysis of the Medicago glycosyltransferase UGT71G1 reveals residues that control regioselectivity for (Iso) flavonoid glycosylation
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DOI：
10.1074/jbc.m605767200
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发表时间：
2006-11-10
影响因子：
4.8
通讯作者：
Dixon, Richard A.
中科院分区：
文献类型：
--
作者：
He, Xian-Zhi;Wang, Xiaoqiang;Dixon, Richard A.
植物糖基转移酶UGT 71 G1从模式豆科桶medic（苜蓿）糖基化类黄酮，甘草，和三萜。它可以将葡萄糖转移到黄酮醇槲皮素的五个羟基中的每一个，其中3 '-O-葡萄糖苷作为主要产物，并转移到异黄酮的A-环7-羟基。糖供体和受体结合口袋分别位于最近确定的UGT 71 G1晶体结构的N和C末端。通过定点诱变系统地改变形成UGT 71 G1的结合口袋的残基。Phe（148）突变为瓦尔，或Tyr（202）突变为Ala，显著改变了槲皮素糖基化的区域选择性，从B环的3 '-O-位主要改变为C环的3-O-位。Y202 A突变体对槲皮素的催化效率与野生型酶相当，而F148 V突变体的效率降低了3-4倍。Y202 A突变体获得了对染料木素5-羟基进行糖基化的能力。额外的突变影响了糖供体UDP-半乳糖和UDP-葡萄糖醛酸的相对特异性，尽管UDP-葡萄糖总是优选的。结果进行了讨论，在新的生物催化剂的生产治疗类黄酮的设计。
The plant glycosyltransferase UGT71G1 from the model legume barrel medic ( Medicago truncatula) glycosylates flavonoids, isoflavonoids, and triterpenes. It can transfer glucose to each of the five hydroxyl groups of the flavonol quercetin, with the 3'-O-glucoside as the major product, and to the A-ring 7-hydroxyl of the isoflavone genistein. The sugar donor and acceptor binding pockets are located in the N and C termini, respectively, of the recently determined crystal structure of UGT71G1. The residues forming the binding pockets of UGT71G1 were systematically altered by site-directed mutagenesis. Mutation of Phe(148) to Val, or Tyr(202) to Ala, drastically changed the regioselectivity for quercetin glycosylation from predominantly the 3'-O-position of the B-ring to the 3-O-position of the C ring. The Y202A mutant exhibited comparable catalytic efficiency with quercetin to the wild-type enzyme, whereas efficiency was reduced 3-4-fold in the F148V mutant. The Y202A mutant gained the ability to glycosylate the 5-hydroxyl of genistein. Additional mutations affected the relative specificities for the sugar donors UDP-galactose and UDP-glucuronic acid, although UDP-glucose was always preferred. The results are discussed in relation to the design of novel biocatalysts for production of therapeutic flavonoids.