Molecular cloning and characterization of caffeic acid 3-O-methyltransferase from the rhizome of Ligusticum chuanxiong

• Published in: Biotechnology letters 2015-11, Vol.37 (11), p.2295-2302
• Main Authors: Li, Juan-Juan, Zhang, Gan, Yu, Ji-hua, Li, Yang-yang, Huang, Xin-he, Wang, Wan-Jun, Tan, Rui, Zhou, Jia-yu, Liao, Hai
• Format: Article
• Language: English
• Subjects: Amino acids; Applied Microbiology; Aquatic plants; Biochemistry; Biomedical and Life Sciences; Biotechnology; caffeate O-methyltransferase; caffeic acid; Chilling; Cloning; Cloning, Molecular; cold treatment; Cooling; E coli; Escherichia coli; Escherichia coli - genetics; Ferulic acid; high performance liquid chromatography; In vitro testing; Life Sciences; Ligusticum; Ligusticum - enzymology; Ligusticum sinense; Liquid chromatography; Medicinal herbs; medicinal plants; Medicine; Methyltransferases - chemistry; Methyltransferases - genetics; Methyltransferases - metabolism; Microbiology; Molecular biology; molecular cloning; nuclear magnetic resonance spectroscopy; open reading frames; Original Research Paper; Pharmaceuticals; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Recombinant; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rhizome - enzymology; rhizomes; traditional medicine
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-015-1917-y

OBJECTIVES: To clone and characterize caffeic acid 3-O-methyltransferase (LcCOMT) from the rhizome of Ligusticum chuanxiong, a traditional medicinal herb having a high content of ferulic acid. RESULTS: LcCOMT encoded an ORF of 362 amino acids with a calculated MW of 39,935 Da and pI of 5.94. Polygenetic tree indicated that LcCOMT was attributed to a new member of COMTs in plants. The recombinant LcCOMT was expressed in E. coli. HPLC and ¹H NMR analyses of purified LcCOMT protein confirmed that it could catalyze caffeic acid to produce ferulic acid in vitro. The further site-mutagenesis proved that His268 was one key catalytic residue. In addition, the substantial changing expression level of LcCOMT under chilling treatment suggested that LcCOMT might play important role in the accumulation of ferulic acid under chilling treatment. CONCLUSIONS: This is the first report of the isolation and characterization of a COMT clone from traditional medicine containing high contents of pharmaceutical ferulic acid.