Characterization of the selected haloalkane dehalogenases crystals specific activity towards to their substrates

Katsiaryna Tratsiak ^1,2,Oksana Degtjarik ^1,2, Ivana Drienovska ³, Lukas Chrast ³, Jiri Damborsky ³, Pavlina Rezacova ^4,5, Michal Kuty ^1,6, Radka Chaloupkova ³, Jose A. Gavira ⁷ and Ivana Kuta Smatanova ^1,6.

¹University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, CENAKVA and School of Complex Systems, Zamek 136, 373 33 Nove Hrady

²University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 31, 370 05 České Budějovice, Czech Republic

³Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic

⁴Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, Prague 4, Czech Republic

⁵Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i., Flemingovo nam. 2, 166 37 Prague, Czech Republic

⁶Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady, Czech Republic

⁷Laboratory for Crystallographic Studies (CSIC-UGR) University of Granada, Av. de las Palmeras, 4, 18100 Armilla - Granada, Spain

E-mail: tratsiak@frov.jcu.cz

Haloalkane dehalogenases (EC 3.8.1.5; HLDs) are microbial enzymes with catalytic activity for the hydrolytic conversion of xenobiotic and highly toxic halogenated aliphatic compounds to the corresponding alcohols (J. Damborsky et al, 2001, Janssen et al. 2005). These enzymes are able to convert a wide spectrum of substrates including halogenated alkanes, cycloalkanes, alkenes, ethers, alcohols, ketones, and cyclic dienes (E. Chovancova et al.2007).

Enzymes DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB 17 were used to obtain crystals in optimized crystallization conditions for carrying out further HLD activity reaction according to the Iwasaki method which is based on observing the intensity of color of a dark red iron-thiocyanate complex which is proportional to the concentration of halogen ions in the sample. Crystal of DpcA enzyme diffracted to the 1.05 Å resolution and belonged to the primitive monoclinic space group P2₁ (4), crystals of DmxA diffracted to the resolution of 2.49 Å belonged to centered orthorhombic C222₁ (20) space group were cross-linked by 8% gluteraldehyde and specific activity towards to 1-bromoheaxane and 1,2-dichloroethane at different time were examined.

The research of the represented data was supported by GA CR (P207/12/0775) and ME CR (CZ.1.05/2.1.00/01.0024).