THERMODYNAMIC ANALYSIS OF THE ENANTIOSELECTIVITY OF HALOALKANE DEHALOGENASE DbjA AND ITS VARIANTS DbjAΔ AND DbjAΔ+H139A TOWARDS BROMINATED ESTERS AND β-SUBSTITUTED BROMOALKANES

R. Chaloupkova¹, Z. Prokop¹, T. Mozga¹, Y. Sato², T. Koudelakova¹, Y. Nagata², T. Senda³ and J.Damborsky¹

¹Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic; ²Department of Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan; ³Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan

Haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 [1] shows excellent enantioselectivity towards brominated esters (E-value > 200) and high enantioselectivity towards b-substituted bromoalkanes (E-value ≤ 145). Substrate mapping revealed that brominated esters are kinetically resolved by DbjA as well as two other haloalkane dehalogenases DhaA Rhodococcus rhodochrous NCIMB13064 and LinB from Sphingobium japonicum UT26, while b-substituted bromoalkanes only by DbjA.

Structural analysis and comparison of the primary sequence of DbjA with protein sequences of other family members revealed the presence of an inserted fragment unique to DbjA, which is located on the protein surface. Construction and characterization of DbjAΔ variant carrying deletion of the extra amino acid sequence showed that the fragment is involved in enantioselectivity of DbjA with b-substituted bromoalkanes, but not with the brominated esters. Following detailed comparison of crystal structures of DbjA and DbjAΔ enzymes indicate importance of amino acid residue H139 to chiral recognition of b-substituted bromoalkanes, resulting in the construction of DbjAΔ+H139A variant. Its kinetic characterization revealed modulation of enantioselectivity towards β-substituted bromoalkanes and no changes in enantioselectivity with brominated esters.

In this work, the thermodynamic analysis has been used to address the origin of enantiomeric discrimination by determining differential activation enthalpy and entropy for the reaction of DbjA, DbjAΔ and DbjAΔ+H139A enzyme variants with selected brominated esters and b-substituted bromoalkanes. Differential activation enthalpy was found to be a major determinant of chiral recognition for brominated esters by all three enzyme variants. Enzyme enantioselectivity towards brominated esters could be explained by different binding interactions of individual enantiomers with the residues of the active site in the Michaelis complex and/or the transition state of dehalogenation reaction. On the other hand, entropy was found to play equally important role as enthalpy for enantiomeric discrimination of 2-bromopentane by DbjA and DbjAΔ+H139A. Interestingly, DbjAΔ showed reversed dependence of enantioselectivity on temperature for 2-bromopentane when entropy dominated over enthalpy contribution towards enzyme enantioselectivity. Importance of entropy for kinetic resolution of β-substituted bromoalkanes by all studied enzymes can be related to different flexibility of both enantiomers in the enzyme active site and different solvation/desolvation of the enzyme active site upon binding.

In summary, obtained results imply that DbjA possesses two chemically distinct bases of enantioselectivity towards brominated esters and β-substituted bromoalkanes and that enzyme variants with different thermodynamic contributions towards enantioselectivity can be constructed by protein engineering.

[1] Sato, Y., Monincova, M., Chaloupkova, R., Prokop, Z., Ohtsubo, Y., Minamisawa, K., Tsuda, M., Damborský, J., and Nagata, Y. (2005) Appl. Environ. Microbiol. 71, 4372-4379