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

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 Km 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.