Structure-enantioselectivity relationships of haloalkane dehalogenases
Tomáš Mozga1, Zbyněk Prokop1, Yukari Sato2, Petr Jeřábek1, Yuji Nagata2, Toshiya Senda3, Jiří Damborský1
Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4,
625 00 Brno, Czech Republic
2 Department of Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan
3 Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan
Haloalkane dehalogenases (EC 22.214.171.124) are enzymes able to remove a halide from halogenated aliphatic compounds by the hydrolytic replacement. Products of this reaction are corresponding alcohols. Recently we have demonstrated for the first time that certain proteins from this enzymatic family possess sufficient enantioselectivity for synthesis of optically active compounds. There is a high potential for an application of haloalkane dehalogenases in asymmetric biocatalysis, since optically active haloalkanes and alcohols are valuable building blocks in organic synthesis of intermediates for pharmaceuticals, agrochemicals, food additives, and odorants.
Hydrolytic dehalogenation of wide range of racemic substrates catalysed by haloalkane dehalogenases DhaA from Rhodococcus rhodochrous NCIMB 13064, LinB from Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 and DbjA from Bradyrhizobium japonicum USDA 110 have been tested in this study. Three different haloalkane dehalogenases posses excellent enantioselectivity with brominated esters (E-value > 200). Additionally, the haloalkane dehalogenase DbjA revealed high enantioselectivity towards b-substituted bromoalkanes. This enantioselectivity is interesting due to the simple structure of these chemical compounds giving a very few possibilities for different binding and catalysis of (R) and (S) enantiomers. Sequence comparisons and structural analysis revealed the presence of insertion fragment unique to DbjA that is located on protein surface. Mutagenesis followed by kinetic characterization of deletion mutant confirmed that this fragment is involved in enantioselectivity of DbjA with b-substituted bromoalkanes, but not with esters. This evidence suggests that DbjA enzyme is acting by two different enantioselectivity mechanisms. This study demonstrates that enatioselectivity of enzymes can be modulated by the engineering of surface loop which may have important implications for construction of new enantioselective biocatalysts.