Pu Gaobin, Wang Peng, Zhou Bingqian, Liu Zhenhua, Xiang Fengning
Key Laboratory of Plant-Cell Engineering and Germ-plasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Biosci Biotechnol Biochem. 2013;77(7):1403-9. doi: 10.1271/bbb.130011. Epub 2013 Jul 7.
Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance.
金银花在中国医学中被用作抗氧化剂的来源，主要是黄酮类化合物，以及一种酚酸绿原酸（CGA）。在此，我们报告了LjC3H全长cDNA的分离和表征，LjC3H是一个编码对香豆酰酯3-羟化酶的基因，该酶参与CGA的合成。系统发育分析表明，其蛋白质属于CYP98A亚家族，同源建模显示其结构与其他细胞色素P450家族蛋白质的结构相似。Southern印迹分析表明，金银花基因组中存在多个与LjC3H同源的序列拷贝。LjC3H cDNA在大肠杆菌中的异源表达使得能够对LjC3H进行体外测定。该实验表明，该酶更倾向于以对香豆酰莽草酸而非对香豆酰奎尼酸作为底物。用茉莉酸甲酯处理叶片以及暴露于UV-B辐射均会增加LjC3H转录本丰度。金银花叶片中的CGA水平与LjC3H转录本丰度呈正相关。
