Molecular Docking and Site-Directed Mutagenesis of Dichloromethane Dehalogenase to Improve Enzyme Activity for Dichloromethane Degradation

Dichloromethane (DCM) dehalogenase in bacterial cells can catalyze the degradation of deleterious DCM in environments. However, the utility of naturally occurring DCM dehalogenase is often limited due to low enzyme activity and content in living cells. In this study, the gene encoding DCM dehalogenase was cloned from Methylobacterium rhodesianum and overexpressed in Escherichia coli. Based on molecular docking analysis of DCM dehalogenase using DCM as the ligand, all of the target amino acid residues within substrate binding pocket and 10 conservative amino acid residues were individually mutated to Ala. After determination of activity, R120, L121, W128, and T146 were chosen for further saturation mutation. Results showed that dcmT146A, dcmT146R, and dcmT146Q have higher activities, whereas dcmL121A, dcmT146L, dcmL121Q, and dcmL121F have retained activities. Next, these seven mutants with a single mutation on amino acid residue were chosen for double mutation. It was found that the mutant of dcmL121A/T146R exhibits the highest activity increasing by 52.8% relative to wild type. Bioinformatic and experimental analyses revealed that the mutant variant dcmL121A/T146R bears the reduced steric hindrance in the active center with a decreased number of amino acid residues within binding pocket from 8 to 5 while overall hydrophilicity increased. In addition, the number of hydrophobic amino acid residues within substrate binding pocket increased while K-m value decreased. It was speculated that all these changes in mutant variant dcmL121A/T146R may contribute to the increase in catalytic activity. It can be concluded that our goal-orientated manipulation through homology modeling, molecular docking, and site-directed mutagenesis is effective for improvement of DCM dehalogenase activity and investigation of correlation between structure and function.