Structure-functional characterization of the member of α/β- hydrolases superfamily - DpcA from Psychrobacter cryohalolentis K5

Katsiaryna Tratsiak 1*, Ivana Drienovska 2, Pavlina Rezacova 3,4, Radka Chaloupkova 2, Michal Kuty 1,5, Jose A. Gavira6 , Oksana Degtjarik1,7, Jiri Damborsky 2  and Ivana Kuta Smatanova 1,5

 

1 Faculty of Science, University of South Bohemia in Ceske Budejovice Budweis, Czech Republic

2 Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University  Brno, Czech Republic

3 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Prague , Czech Republic

4 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i.,  Prague, Czech Republic

5 Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Nove Hrady, Czech Republic

6Laboratory for Crystallographic Studies (CSIC-UGR) University of Granada, Granada, Spain

7 Weizmann Institute of Science, Department of Structural Biology, Rehovot, Israel 

ktratsiak@gmail.com

 

The magnification of the anthropogenic influences of different pollutants on the biosphere, for instance halogenated alkanes, necessitate the searching of new methods and resources for their decontamination and degradation. Haloalkane dehalogenase (EC 3.8.1.5; HLDs) DpcA isolated from psychrophilic Psychrobacter cryohalolentis K5 is aimed on the solving of the issue mentioned above by catalysing the hydrolytic conversion of halogenated aliphatic compounds accomplished by cleavage of carbon-halogen bond with the subsequent releasing of the corresponding alcohol, halide ion and a proton.

DpcA has high enantioselectivity and the narrowest substrate specificity of all biochemically characterized HLDs with the highest activity of the enzyme toward 1-bromobutane and 1,3-dibromopropane at 25 °C what highlights it among the other HLDs.

The crystals of DpcA diffracted to the resolution 1.05 Å beamline 14.2, detector Rayonics MX-225 CCD (BESSY II electron-storage ring, HZB, Germany), belonged to P21 space group and with one molecule in the asymmetric unit. The structure was solved by molecular replacement with the help of the coordinates of Xanthobacter autotrophicus (PDB code: 1B6G; 40% sequence identities).

The protein has a globular shape and is composed of two domains. The core domain contains eight parallel β-strands that form central beta-sheet, within one is antiparallel (β2). The central β-sheet is flanked by four α-helices on the one side and two are on the other side of the sheet. The highly conserved main domain is the scaffold- like for the catalytic residues, and a smaller helical cap domain, covering the active site, which has revealed the catalytic pentad essential for for the SN2 reaction mechanism: nucleophile D123, catalytic base H280, catalytic acid D250, halide-stabilizing W124 and W164. 

For the structure analysis the crystals were soaked with the ligands: 1-bromohexane, 1-bromobutane, 1,2-dibromoethane and 2-bromopropane. The comparison of structure of DpcA and DpcA with 1,2-dibromoethane is carried out for the understanding the psychrophilic properties of the enzyme. 

We thank Manfred Weiss and Sandra Pühringer for their assistance with data collection at the MX 14.2 BESSY beamline in Berlin. This work is supported by the Grant Agency of the Czech Republic (P207/12/0775). Also was supported by the Ministry of Education of the Czech Republic (CZ.1.05/2.1.00/01.0024 and CZ.1.05/2.1.00/01.0001). The support of the Academy of Sciences of the Czech Republic is acknowledged as well.

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Keywords: α/β- hydrolases  haloalkanes  molecular replacement  enzyme structure