Deciphering Reductive Dehalogenase Specificity Through Targeted Mutagenesis of Chloroalkane Reductases
Reductive dehalogenases (RDases) are essential in the anaerobic degradation of various organohalide contaminants. This family of enzymes has broad sequence diversity, but high structural conservation. There have been few studies assessing how RDase peptide sequences affect their substrate selectivity. Here we focus on two chloroalkane RDases, CfrA and DcrA, which have 95% protein sequence identity but have diverged to hold distinct substrate preferences. CfrA will dechlorinate chloroform and 1,1,1-trichloroethane, whilst DcrA will dechlorinate 1,1-dichloroethane. We mutated several residues in the active site of CfrA to investigate a change in substrate preference and to identify which wild-type residues contribute the most to substrate specialization. We determined that no individual residue solely dictates substrate discrimination, but both Y80W and F125W mutations were needed to force CfrA to prefer 1,1-dichloroethane as a substrate. This double mutation also altered the transformation pathway of 1,1,2-trichloroethane from hydrogenolysis (forms 1,2-dichloroethane) to dihaloelimination (forms vinyl chloride). We use predictive protein models and substrate docking to predict what interactions are made between the enzyme and substrate to aid in selection. The residues of significance identified in this study are consistent with those identified from chloroethene RDases, suggesting residue locations with a particularly high impact on activity. ImportanceReductive dehalogenases play an integral role in the removal of chlorinated solvents from the environment. These enzymes have specificity towards different chlorinated compounds, and it is known that small natural changes in their peptide sequence can change their activity drastically. How these specific sequence variations influence activity is largely unknown. In this study, we demonstrate that mutating a few residues within the active site of CfrA--a chloroform and trichloroethane-specific dehalogenase--changes its substrate preference to dichloroethane. We determine that only two mutations are needed to disrupt the native activity, underscoring the nuances in substrate-structure relationships in reductive dehalogenases. Though we are still far from predicting function from the sequence, this knowledge can give some insight into engineering reductive dehalogenases for new target contaminants.