From Energy Deprivation Stress to Catalytic Adaptation of Reductive Dehalogenase TmrA for 1,1‐ DCA Dechlorination: An Experimental and in Silico Study

Organohalide-respiring bacteria capable of metabolizing multiple organohalogens represent valuable tools for bioremediation and offer intriguing evolutionary potential for adaptation to non-native substrates. In the present study, a chloroform respiring Dehalobacter restrictus strain was examined for its adaptability to a low affinity substrate (1,1-dichloroethane) over five subcultures (~28 generations). We obtained an enhanced 11,1-dichloroethane dechlorination rate by culturing with a 1:9 ratio of native (chloroform) to non-native (1,1-dichloroethane) substrates. We identified mutations corresponding to amino acids located in high-entropy regions of the TmrA protein sequence, suggesting mutational plasticity at these sites. We revealed that the mutated TmrA structure showed increased binding affinity for 1,1-dichloroethane by using molecular dynamics simulations and binding free energy computations. These findings provide insights into the adaptability of anaerobic organohalide-respiring bacteria toward non-native organohalogens and identify structural features of reductive dehalogenases that may be exploited for future enzyme engineering in bioremediation applications.