Zhang Yue, Lv Hai-peng, Ma Cheng-ying, Guo Li, Tan Jun-feng, Peng Qun-hua, Lin Zhi
Key Laboratory of Tea Biology and Resource Utilization of Ministry of Agriculture, Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China.
J Zhejiang Univ Sci B. 2015 Feb;16(2):103-12. doi: 10.1631/jzus.B1400193.
Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) present in leaves of Camellia sinensis has many beneficial biological activities for human health. However, EGCG3"Me occurs naturally in tea leaves in extremely limited quantities. Finding an enzyme from C. sinensis to catalyze the synthesis of EGCG3"Me is an alternative method to make up for the scarcity of EGCG3"Me in natural situations. In the present study, a complementary DNA (cDNA) encoding region and genomic DNA of the caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) gene were isolated from C. sinensis (designated CsCCoAOMT). Nucleotide sequence analysis of CsCCoAOMT revealed an open reading frame of 738 bp that encodes a polypeptide with a predicted molecular weight of 28 kDa, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The full-length DNA sequence (2678 bp) contained five exons and four introns. The deduced amino acid sequence of CsCCoAOMT shared 92% identity with CCoAOMTs from Codonopsis lanceolata and Betula luminifera. The catalytic activity of CsCCoAOMT was analyzed. Three monomethylated epigallocatechin-3-O-gallate (EGCG) compounds (EGCG4"Me, EGCG3"Me, and EGCG3'Me) were produced by CsCCoAOMT with K(m) in the micromolar range. Real-time polymerase chain reaction (RT-PCR) experiments indicated that the CsCCoAOMT transcript was present at low levels during the early stages of leaf maturity (the first leaf and bud on a shoot) but the relative expression was augmented at advanced stages of leaf maturity (the third or fourth leaf on a shoot), which accorded well with changes in EGCG3"Me content in fresh leaves. Hence, we concluded that CsCCoAOMT catalyzes the syntheses of methylated EGCGs.
茶树叶片中含有的表没食子儿茶素-3-O-(3-O-甲基)没食子酸酯（EGCG3″Me）对人体健康具有多种有益的生物活性。然而，EGCG3″Me在茶叶中的天然含量极其有限。从茶树中寻找一种酶来催化EGCG3″Me的合成是弥补自然条件下EGCG3″Me稀缺的一种替代方法。在本研究中，从茶树中分离出了编码咖啡酰辅酶A O-甲基转移酶（CCoAOMT）基因的互补DNA（cDNA）编码区和基因组DNA（命名为CsCCoAOMT）。对CsCCoAOMT的核苷酸序列分析显示，其开放阅读框为738 bp，编码一个预测分子量为28 kDa的多肽，这与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）的结果高度相关。全长DNA序列（2678 bp）包含5个外显子和4个内含子。CsCCoAOMT推导的氨基酸序列与轮叶党参和光皮桦的CCoAOMT有92%的同一性。分析了CsCCoAOMT的催化活性。CsCCoAOMT产生了三种单甲基化的表没食子儿茶素-3-O-没食子酸酯（EGCG）化合物（EGCG4″Me、EGCG3″Me和EGCG3′Me），其米氏常数（K(m)）在微摩尔范围内。实时聚合酶链反应（RT-PCR）实验表明，CsCCoAOMT转录本在叶片成熟早期（嫩梢上的第一片叶子和芽）含量较低，但在叶片成熟后期（嫩梢上的第三或第四片叶子）相对表达量增加，这与鲜叶中EGCG3″Me含量的变化高度一致。因此，我们得出结论，CsCCoAOMT催化甲基化EGCG的合成。
