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
Rezacova3,4, Michal Kuty1,5, Radka
Chaloupkova2, and Ivana Kuta Smatanova1,5.
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.