Homology modeling and site-directed mutagenesis of caffeic acid-3-O-methyltransferase from Ligusticum chuanxiong / 中草药

ABSTRACT

Objective: To construct the three dimensional models of L. chuanxiong caffeic acid-3-O-methyltransferase (LCCOMT) and verify the model using site-directed mutagenesis technology. Methods: The three-dimensional model was constructed by homology modeling using the crystal structure of COMT from Medicago sativa as a temple. Caffeic acid was docked into the optimized model of LCCOMT to predict the active site. The predicted site was mutated using site-directed mutagenesis technology. Then, the activity of mutant enzyme was detected. Results: The molecular docking, which showed there were hydrogen bonds between His268 and 3-OH of caffeic acid, was successful. Two mutant enzymes, H268N and H268Q, lost 94.85% and 95.28% of their activity respectively. Conclusion: The His268 is confirmed as one of the key residues of LCCOMT. It may play a role as a base in the deprotonation reaction of the 3-OH of caffeic acid.