Stereoselectivity and Conformational Stability of Haloalkane Dehalogenase DbjA from Bradyrhizobium japonicum USDA110: The Effect of pH and Temperature
Chaloupkova, R., Prokop, Z., Sato, Y., Nagata, Y., Damborsky, J.
FEBS JOURNAL 278: 2728-2738 (2011)
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 range of 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 was distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stability. 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 at the pH interval of 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, ΔR-SΔH‡ and ΔR-SΔS‡, 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 mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA at a broad pH range, combined with high enantioselectivity with particular substrates, makes this enzyme a very versatile biocatalyst.