Yuan Yuan, Yu Shulin, Yu Jun, Zhan Zhilai, Li Minhui, Liu Guiming, Wang Xumin, Huang Luqi
State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, Academy of Chinese Medical Sciences, Beijing 100700, China.
Pharmacy College, Anhui University of Chinese Medicine, Hefei 230038, China.
Int J Mol Sci. 2014 Feb 10;15(2):2386-99. doi: 10.3390/ijms15022386.
4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1.
4-香豆酸：辅酶A连接酶（4CLs）是参与苯丙烷类衍生化合物代谢途径的一组关键酶；然而，仅基于保守结构域的序列相似性来鉴定这些重要酶的直系同源物和旁系同源物仍然很困难。利用来自公共数据库的20种植物物种的序列数据以及忍冬的序列，我们定义了1252个依赖于腺苷单磷酸（AMP）的合成酶/连接酶序列，并将它们分为三个系统发育分支。根据它们的分类，4CLs属于四个亚组之一，其中已知的蛋白质在拟南芥和水稻中有特征描述。我们还定义了184个非冗余序列，这些序列编码含有GEICIRG基序的蛋白质，并且这些含GEICIRG蛋白质的分类分布表明植物中存在独特的催化活性。我们进一步分析了它们在忍冬和忍冬变种金银花中的转录水平，并选择了代表亚组的最高表达基因进行结构和结合位点预测。结合忍冬花的液相色谱-质谱（LC-MS）分析，对LJ4CL1保守结合位点的假定底物结合氨基酸残基、阿魏酸和4-香豆酸的结构研究得出结论，该花中高表达的蛋白质组可能处理占已知苯丙烷类衍生化合物90%的4-香豆酸。以4-香豆酸为模板分析纯化的粗LJ4CL1蛋白的活性，高活性表明4-香豆酸是LJ4CL1的底物之一。
