Crystallization and structural characterization of LinB86 haloalkane dehalogenase mutant

O. Degtjarik^1,2, I. Iermak^1,2, J.  Rezabek³, R. Chaloupkova⁵, P. Rezacova⁴, M. Kuty^2,3, J. Damborsky⁵ and I. Kuta Smatanova^1,3

¹University of South Bohemia, Faculty of Science, Branišovska 31, CZ-37005 Česke Budejovice, Czech Republic

²Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady, Czech Republic

³University of South Bohemia, Faculty of Fisheries and Protection of Waters, Institute of Complex Systems and CENAKVA, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic

⁴Institute of Molecular Genetics of the Academy of Science of the Czech Republic, v.v.i., Flemingovo no. 2, 16637 Prague, Czech Republic

⁵Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

LinB is a microbial enzyme of the haloalkane dehalogenase family that catalyze the cleavage of the carbon-halogen bond in halogenated aliphatic pollutants, resulting in the formation of a corresponding alcohol, a halide ion and a proton. Haloalkane dehalogenase LinB isolated from a bacterium Sphingobium japonicum UT26 has relatively broad substrate specificity and can be potentially used for biosensing and biodegradation of environmental pollutants. Different variants of haloalkane dehalogenase LinB were constructed with a goal to study the effect of mutations on enzyme functions. In LinB86 (W140A+F143L+L177W+I211L) variant mutations lead to blocking of the main tunnel and opening of alternative way for connection the deeply buried active site with the surrounding solvent.

Crystals of LinB86 were obtained Index (Hampton Research, USA) and Morpheus (Molecular Dimensions Ltd., UK) screens. Full data set was collected on the beamline ID-29 of ESRF (Grenoble, France) at 2.43 Å resolution. Crystals belong to H-centered trigonal H32 space group. Matthews coefficient of V_M = 2.55 ų Da^-1 suggests that the crystals contain two molecules per asymmetric unit, which corresponds to a solvent content of 51.79%. Crystal structure of LinB86 was solved by molecular replacement using the coordinates from LinB32 (L177W) haloalkane dehalogenase mutant. The structure refinement is currently in progress.

This research was supported by the GA CR (P207/12/0775), GAJU (141/2013/P) and ME CR (CZ.1.05/2.1.00/ 01.0024).