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.
ABSTRACT
A cDNA clone encoding S-adenosyl-L-methionine: trans-caffeoyl-CoA 3-O-methyltransferase (EC 2·1·1·104; CCoAOMT) from Stellana longipes Goldie (long-stalked chickweed) was isolated and studied. Structural analysis of both the nucleotide sequence and the predicted amino acid sequence suggests that our cloned sequence encoded a CCoAOMT enzyme of Stellaria longipes, which shared overall structural similarity with other plant CCoAOMTs but exhibited certain distinct characteristics. Southern blot hybridization and cloning analyses indicating a small CCoAOMT gene family in the Stellana longipes genome and the absence of introns in the coding region of the cDNA- corresponding gene. Sequence variations in the coding region were found among three genotypes from geographically isolated populations. Higher levels of CCoAOMT mRNA were detected in stems and leaves than in roots. The cDNAencoded protein expressed in Eschendia coli was shown to utilize caffeoyl-CoA, but not caffeic acid or 5-hydroxy ferulic acid, as its substrate.