Haloalkane dehalogenases: DmxA from Marinobacter sp. ELB 17 and DpcA from Psychrobacter cryohalolentis K5, from the crystallization to the structure analysis

Katsiaryna Tratsiak^{* 1}, Tatyana Prudnikova¹, Ivana Drienovska², Lukas Chrast², Jiri Damborsky², Oksana Degtjarik¹, Pavlina Rezacova^3,4, Michal Kuty^1,5, Radka Chaloupkova², and Ivana Kuta Smatanova^1,5.

¹University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 31, 370 05 České Budějovice, Czech Republic, ^*E-mail: ktratsiak@gmail.com

²Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic

³Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic

⁴Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i., Flemingovo nam. 2, 166 37 Prague, Czech Republic

⁵Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady, Czech Republic

The selected enzymes, belonging to the family of haloalkane dehalogenases (EC 3.8.1.5; HLDs), catalyzing the hydrolytic conversion of halogenated aliphatic compounds to their corresponding alcohols, were isolated from Psychrobacter cryohalolentis K5 (DpcA) and from Marinobacter sp. ELB 17 (DmxA).

Both are exctremoenzymes, exhibiting height enantioselectivity, however reveals the highest activity at low and height temperatures, respectively, what highlights them among another HLDs.

The enzymes were crystallized and obtained diffraction data of the crystals was refined to the resolution 1.05 Å for DpcA and 1.45 Å for DmxA. Diffraction data were collected at the beamline 14.2, Helmholtz-Zentrum Berlin (HZB) (Germany) at the BESSY II electron storage ring, equipped with a detector Rayonics MX-225 CCD, wavelength 0.978 Å and on the beamline MX- ID29 at the ESRF electron-storage ring (Grenoble, France), with Pilatus 6M-F detector at the wavelengths of 0.972 Å.

Crystals of DpcA belonged to P2₁ the primitive monoclinic space group with unit-cell parameters: a = 41.3, b = 79.4, c = 43.5 A ˚, α = β = 90.0, γ = 95.0 and contained one molecule in the asymmetric unit. Crystals of DmxA belonged to the primitive orthorhombic P2₁2₁2₁ space group, with unit-cell parameters: a = 43.371, b = 78.343, c = 150.51; α = γ = β = 90.0 and contained 2 molecules in the asymmetric unit. The structures were solved by molecular replacement with MOLREP from the CCP4 software suite. The coordinates of Xanthobacter autotrophicus (PDB code: 1B6G; 40% sequence identities for 121 residues and 53% sequence similarity was used as search model for DpcA structure and for DmxA from Rhodococcus rhodochrous (PDB entry 4E46; 48% sequence identity for 142 residues and 63% sequence similarity). Refining was carried out manually with REFMAC5 and WinCOOT 6.4 from the CCP4 suit.

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.