Two Divergent Members of 4-Coumarate： Coenzyme A Ligase from Salvia miltiorrhiza Bunge： cDNA Cloning and Functional Study

作     者：Shu-Juan Zhao Zhi-Bi Hu DI Liu Frederick C. C. Leungt 

作者机构：Institute of Traditional Chinese Materia Medica Shanghai University of Traditional Chinese Medicine Shanghai 201203 China Department of Zoology University of Hong Kong Hong Kong SAR China 

出 版 物：《Journal of Integrative Plant Biology》 (植物学报（英文版）)

年 卷 期：2006年第48卷第11期

页      面：1355-1364页

核心收录：

学科分类：0710[理学-生物学] 071001[理学-植物学] 07[理学] 

基　　金：Supported by the National Natural Science Foundation of China (30300447). The authors thank Dr Chen Yongning (China Innovation Centre for Drug Development  HK) for useful suggestions and support. The authors also thank to Dr Fanya Zeng and Miss Charis Chan (Department of Zoology  University of Hong Kong) for technical assistance 

主　　题：cDNA cloning 4-coumarate : coenzyme A ligase (4CL) Danshen (Salvia miltiorrhiza) enzymatic kinetic assays water-soluble phenolic acids. 

摘      要：4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.