Abstract

Haloalkane dehalogenases are a very important class of microbial enzymes for environmental detoxification of halogenated pollutants, for biocatalysis, biosensing and molecular tagging. The double mutant (Ile44Leu + Gln102His) of the haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 (DbeAΔCl) was constructed to study the role of the second halide-binding site previously discovered in the wild-type structure. The variant is less active, less stable in the presence of chloride ions and exhibits significantly altered substrate specificity when compared with the DbeAwt. DbeAΔCl was crystallized using the sitting-drop vapour-diffusion procedure with further optimization by the random microseeding technique. The crystal structure of the DbeAΔCl has been determined and refined to the 1.4 Å resolution. The DbeAΔCl crystals belong to monoclinic space group C121. The DbeAΔCl molecular structure was characterized and compared with five known haloalkane dehalogenases selected from the Protein Data Bank.

Highlights

  • Hazardous halogenated compounds are an important class of environmental pollutants.An obvious critical step in the potential biodegradation pathway is the dehalogenation process [1,2].Haloalkane dehalogenases (HLDs) play an essential role in biodegradation of the halogenated pollutants.HLDs are predominantly bacterial enzymes that belong to the superfamily of α/β-hydrolases and catalyze the hydrolytic conversion of a wide range of halogenated aliphatic compounds, and play an important role in bioremediation [3] and industrial biocatalytic processes [4]

  • The tertiary structures of HLDs are composed of a conserved α/β-hydrolase core domain and an α-helical cap domain [5]

  • In order to overexpress DbeA∆Cl in E. coli BL21(DE3) cells, the final genes were transcribed by T7 RNA polymerase, which is expressed by the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible lac UV5 promoter

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Summary

Introduction

Hazardous halogenated compounds are an important class of environmental pollutants.An obvious critical step in the potential biodegradation pathway is the dehalogenation process [1,2].Haloalkane dehalogenases (HLDs) play an essential role in biodegradation of the halogenated pollutants.HLDs are predominantly bacterial enzymes that belong to the superfamily of α/β-hydrolases and catalyze the hydrolytic conversion of a wide range of halogenated aliphatic compounds, and play an important role in bioremediation [3] and industrial biocatalytic processes [4]. Hazardous halogenated compounds are an important class of environmental pollutants. An obvious critical step in the potential biodegradation pathway is the dehalogenation process [1,2]. Haloalkane dehalogenases (HLDs) play an essential role in biodegradation of the halogenated pollutants. HLDs are predominantly bacterial enzymes that belong to the superfamily of α/β-hydrolases and catalyze the hydrolytic conversion of a wide range of halogenated aliphatic compounds, and play an important role in bioremediation [3] and industrial biocatalytic processes [4]. A halide and a hydrogen cation are released during the enzymatic dehalogenation of haloalkanes by HLDs. The tertiary structures of HLDs are composed of a conserved α/β-hydrolase core domain and an α-helical cap domain [5]. The core domain is responsible for the catalytic reaction of the enzyme

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