Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity

茶树中咖啡酰辅酶A氧甲基转移酶的基因克隆及其催化活性研究(英文)

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（c DNA） encoding region and genomic DNA of the caffeoyl-coenzyme A O-methyltransferase（CCo AOMT） gene were isolated from C. sinensis（designated Cs CCo AOMT）. Nucleotide sequence analysis of Cs CCo AOMT revealed an open reading frame of 738 bp that encodes a polypeptide with a predicted molecular weight of 28 k Da, 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 Cs CCo AOMT shared 92% identity with CCo AOMTs from Codonopsis lanceolata and Betula luminifera. The catalytic activity of Cs CCo AOMT was analyzed. Three monomethylated epigallocatechin-3-O-gallate（EGCG） compounds（EGCG4＂Me, EGCG3＂Me, and EGCG3＇Me） were produced by Cs CCo AOMT with K m in the micromolar range. Real-time polymerase chain reaction（RT-PCR） experiments indicated that the Cs CCo AOMT 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 Cs CCo AOMT catalyzes the syntheses of methylated EGCGs.

目的:从茶树中克隆一种可以催化表没食子儿茶素没食子酸酯(EGCG)生成甲基化EGCG的酶——咖啡酰辅酶A氧甲基转移酶(CCo AOMT),实现甲基化EGCG的酶学合成,为甲基化EGCG的进一步开发利用提供理论依据和技术指导。创新点:本研究首次从茶树中克隆了一条CCo AOMT基因组序列;分析了CCo AOMT基因在不同茶树品种和不同成熟度茶鲜叶中的基因表达规律;证明了CCo AOMT具有催化合成甲基化EGCG的生物活性。方法:采用聚合酶链式反应(PCR)和序列分析获得CCo AOMT的编码序列和基因组序列;采用高效液相色谱-四级杆-飞行时间串联质谱技术(HPLC-QTOF-MS)分析酶促反应生成的甲基化EGCG产物(图4);采用实时荧光定量PCR分析CCo AOMT基因的表达差异(图5)。结论:本研究从茶树中克隆了CCo AOMT基因的编码序列(738 bp)和基因组序列(2678 bp),明确了该基因具有4个内含子和5个外显子;揭示了CCo AOMT可以催化EGCG生成EGCG4"Me、EGCG3"Me和EGCG3'Me等多种甲基化产物;证明了CCo AOMT具有催化生成甲基化EGCG的活性;并发现该基因的表达量高低与茶鲜叶的成熟度呈正相关关系。