Background: Haloalkane dehalogenases (EC 220.127.116.11, HLDs) are α/β-hydrolases which
catalyze the irreversible cleavage of carbon-halogen bonds of haloalkanes, producing an alcohol, a
halide and a hydrogen ion. Haloalkanes are acutely toxic to animals and humans and their toxic
effects are mainly observed in the liver, kidneys and central nervous system.
Objective: In the present work, the haloalkane dehalogenase from Rhizobium leguminosarum bv.
trifolii (DrlA) was characterized.
Method: Reverse transcription polymerase chain reaction analysis and enzyme activity assays revealed
that the DrlA gene expression in R. leguminosarum bv. trifolii is induced by 1,2-
dibromoethane (1,2-DBE) during the early exponential phase. The gene of the enzyme was isolated,
cloned and expressed in E. coli Rosetta (DE3).
Results: Recombinant DrlA displays its high catalytic activity towards 1,2-DBE and the long-chain
haloalkane 1-iodohexane. Limited activity was observed for other aliphatic and cyclic haloalkanes,
indicating that the enzyme displays restricted substrate specificity, compared to other bacterial
HLDs. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the
HLD-II subfamily and shares the same overall fold and domain organization as other bacterial
HLDs, however major variations were identified at the hydrophobic substrate-binding cavity, the
cap domain and the entrance of the main tunnel that affect the size of the active site pocket and the
substrate recognition mechanism.
Conclusion: This work sheds new light on the environmental fate and toxicity of 1,2-DBE and
provides new knowledge on the structure, function and diversity of HLDs for developing applications