Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, Villum Foundation Research Centre "Pro-Active Plants", UNIK Center for Synthetic Biology, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Plant J. 2011 Oct;68(2):287-301. doi: 10.1111/j.1365-313X.2011.04695.x. Epub 2011 Aug 19.
Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l-valine and l-isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside-specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2-hydroxy-2-methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co-occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co-expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine-tuning nitrogen assimilation in cassava.
木薯含有两种氰苷葡萄糖苷，分别为亚麻苦苷和卡南醇，由 L-缬氨酸和 L-异亮氨酸生物合成。本研究从木薯中分离出两个尿苷二磷酸糖基转移酶（UGT）的 cDNA 序列，分别命名为 UGT85K4 和 UGT85K5。这两个基因编码的蛋白具有 96%的氨基酸序列一致性，与高粱和甜樱桃中氰苷特异性 UGT 家族同源。在大肠杆菌中表达的重组 UGT85K4 和 UGT85K5 能够使丙酮氰醇和 2-羟基-2-甲基丁腈发生糖基化反应，分别生成亚麻苦苷和卡南醇。UGT85K4 和 UGT85K5 表现出广泛的体外底物特异性，能够使其他羟腈、一些类黄酮和简单醇类化合物发生糖基化反应。免疫组织化学定位研究表明，UGT85K4 和 UGT85K5 与 CYP79D1/D2 和 CYP71E7 基因家族成员共表达，这些基因家族成员在木薯氰苷合成的早期步骤中发挥作用。这些酶均存在于第一片展开的木薯叶的叶肉和木质部薄壁组织细胞中。原位 PCR 结果表明，UGT85K4 和 UGT85K5 与 CYP79D1 以及 CYP71E7 基因家族的两个成员在第一片展开的叶片叶柄的皮层、木质部和韧皮部薄壁组织以及内皮层的特定细胞中共表达。根据获得的数据，UGT85K4 和 UGT85K5 被认为是催化植物体内氰苷葡萄糖苷合成的 UGT。生物合成酶的定位表明，氰苷葡萄糖苷可能在木薯的防御反应和氮素同化的精细调控中发挥作用。
