Xu Rui-Xue, Ni Rong, Gao Shuai, Fu Jie, Xiong Rui-Lin, Zhu Ting-Ting, Lou Hong-Xiang, Cheng Ai-Xia
Key Laboratory of Chemical Biology of Natural Products, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, Shandong, China.
Department of Pharmacy, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China.
Plant Sci. 2022 Jan;314:111102. doi: 10.1016/j.plantsci.2021.111102. Epub 2021 Oct 25.
Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.
咖啡酰辅酶A O-甲基转移酶（CCoAOMT）催化S-腺苷甲硫氨酸的甲基转移至咖啡酰辅酶A的羟基部分，这是木质素生物合成途径的一部分。类CCoAOMT蛋白也催化多种黄酮类化合物、香豆素和苯丙烷类化合物的反应。从地钱中已鉴定出几种以黄酮类化合物为底物的CCoAOMT。在此，我们从地钱苍白苔（Marchantia paleacea）中克隆了两个CCoAOMT基因，即MpalOMT2和MpalOMT3。MpalOMT3下游有第二个ATG密码子，缺失11个氨基酸的截短版本被命名为MpalOMT3-Tr。系统发育分析表明，MpalOMT3位于维管植物真正的CCoAOMT所在进化枝的根部，而MpalOMT2位于CCoAOMT和类CCoAOMT蛋白之间。重组OMT使咖啡酰辅酶A、苯丙烷类化合物以及含有两个或三个相邻羟基的黄酮类化合物发生甲基化。MpalOMT3对苯丙烷类化合物的催化活性高于MpalOMT2，但MpalOMT2对圣草酚和杨梅素的催化作用更为多样。拟南芥茎中的木质素含量随着MpalOMT3-Tr的组成型异源表达而增加，但MpalOMT2则不然。亚细胞定位实验表明，MpalOMT3的N端可能作为叶绿体转运肽并抑制其酶活性。结合系统发育分析和功能表征，我们得出结论，地钱苍白苔含有真正的CCoAOMT和类CCoAOMT基因。
