Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: The effect of pH and temperature

Radka Chaloupkova, Zbynek Prokop, Yukari Sato, Yuji Nagata, Jiri Damborsky

Research output: Contribution to journalReview articlepeer-review

23 Citations (Scopus)


The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (Tm = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters and were determined. The thermodynamic analysis revealed that the resolution of β-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of α-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst.

Original languageEnglish
Pages (from-to)2728-2738
Number of pages11
JournalFEBS Journal
Issue number15
Publication statusPublished - 2011 Aug


  • activity
  • enantioselectivity
  • haloalkane dehalogenase
  • oligomerization
  • pH
  • structure
  • thermostability


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