Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites

Radka Chaloupkova, Tatyana Prudnikova, Pavlina Rezacova, Zbynek Prokop, Tana Koudelakova, Lukas Daniel, Jan Brezovsky, Wakako Ikeda-Ohtsubo, Yukari Sato, Michal Kuty, Yuji Nagata, Ivana Kuta Smatanova, Jiri Damborsky

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)


The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.

Original languageEnglish
Pages (from-to)1884-1897
Number of pages14
JournalActa Crystallographica Section D: Biological Crystallography
Issue number7
Publication statusPublished - 2014 Jul


  • catalytic activity
  • enzyme stability
  • halide-binding site
  • haloalkane dehalogenase
  • substrate specificity


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