Crystallisation and structure-functional analysis of a novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94

 

Tatyana Prudnikovaa, Tomas Mozgac, Pavlina Rezacovae,f, Radka Chaloupkovac, Yukari Satog, Michal Kutya,b, Tana Koudelakovac, Yuji Nagatag, Jiri Damborskyc,d, and Ivana Kuta Smatanovaa,b

 

aInstitute of Physical Biology University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic

bInstitute of Systems Biology and Ecology Academy of Science of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

cLoschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic

dLoschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic

eInstitute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague, Czech Republic

fInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague, Czech Republic

gDepartment of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan

 

 

A novel enzyme, DbeA, belonging to the family of haloalkane dehalogenases (EC 3.8.1.5) was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to DbjA enzyme from Bradyrhizobium japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. To understand the altered activity and specificity of DbeA enzyme, its mutant variant DbeA1, carrying the unique fragment of DbjA, was also constructed. Both the wild type DbeA and the DbeA1 were crystallised using the sitting-drop vapour-diffusion method. The crystals of DbeA belong to the primitive orthorhombic space group P212121, while the crystals of the mutant DbeA1 belong to the monoclinic space group C2. Crystal structure of a DbeA and DbeA1 has been solved and refined to 2.2 resolution. The enzymatic molecular structure of DbeA was compared with those of known haloalkane dehalogenases already deposited in Brookhaven Protein Data Bank.