Contrasting Kinetics of Highly Similar Chloroalkane Reductive Dehalogenases

Abstract

Chloroform and trichloroethanes are pervasive groundwater contaminants for which bioremediation has been an effective treatment strategy. Reductive dehalogenase (RDase) enzymes from organohalide-respiring bacteria are essential for their remediation under anaerobic conditions. RDases are responsible for dehalogenating these chlorinated solvents, leading to their removal. This work explores the kinetic characteristics of three closely related Dehalobacter chloroalkane-reductases—TmrA, CfrA, and AcdA—and identifies differences between their activity on chloroform (CF), 1,1,1-trichloroethane (TCA), and 1,1,2-TCA. The side-by-side comparison of these enzymes has emphasized that TmrA and AcdA are specialized toward CF with both having 4-fold higher maximum specific activity ( V max ) on CF than 1,1,1-TCA, whereas, CfrA has very similar rates on both CF and 1,1,1-TCA. AcdA is the most sensitive to substrate inhibition by CF and 1,1,2-TCA, and inhibition by a common co-contaminant trichloroethene. Finally, the reduction of 1,1,2-TCA, which can produce both 1,2-dichloroethane and vinyl chloride, was assessed for each enzyme. Interestingly, each enzyme has a distinct preference for the major product it produces, indicating a favoured reaction pathway. Despite over 95% sequence identity, TmrA, CfrA, and AcdA exhibit substantial differences in kinetic behaviour, highlighting the importance of understanding such nuances for informed bioremediation strategies. SYNOPSIS: Three similar dechlorinating enzymes were found to have distinct reaction rates and levels of sensitivity to inhibition. These differences have implications for the enzymes’ use in bioremediation.