Crystallographic study of haloalkane dehalogenase DpcA from Psychrobacter cryohalolentis K5

Katsiaryna Tratsiak^1,2*, Oksana Degtjarik^1,2,6, Tatiana Prudnikova¹, Ivana Drienovska³, Lukas Chrast³, Pavlina Rezacova^4,5, Michal Kuty^1,2,6, Radka Chaloupkova³, Jiri Damborsky³ and Ivana Kuta Smatanova^1,2,6

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

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

³ Loschmidt 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

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

⁵ Institute 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

^*E-mail: tratsiak@frov.jcu.cz

Introduction

Haloalkane dehalogenases (EC 3.8.1.5; HLDs) are microbial enzymes that catalyze the hydrolytic conversion of halogenated aliphatic compounds to their corresponding alcohols [1, 2], which is the hydrolytic dehalogenation accomplished by these enzymes is one of the most important steps in the biodegradation of 1-halo-n-alkanes and α,ω-dihalo-n-alkanes, serious halogenated pollutants [3]. HLDs have a broad substrate specificity [4] and a high enantioselectivity [5], which makes these enzymes applicable in bioremediation [6], in biosensing [7,8], biocatalysis [5, 9], cellular imaging, and protein analysis [10, 11] . Understanding of the structural bases of the enzyme extremophilicity allows for the construction of HLD variants with improved activity and stability at low and high temperatures and thus enlarges their applicability in environmental and biosynthetic applications.

Experimental details

A novel HLD enzyme, DpcA, exhibiting unique temperature profiles with exceptionally high activities at low temperature, isolated from Gram-negative psychrophilic bacteria Psychrobacter cryohalolentis K5 [12] was crystallized by sitting-drop and hanging-drop vapour-diffusion techniques. Crystallization drops were prepared by mixing 2 µl of protein solution at the concentration 10 mg ml^-1 in 50 mM Tris–HCl buffer pH 7.5 and 1 µl precipitant solution plus 0.3–0.6 µl of 0.1 M L-proline. Diffraction data were collected at the beamline 14.2, Helmholtz-Zentrum Berlin (HZB) (Germany) at the BESSY II electron storage ring, detector Rayonics MX-225 CCD [13] at wavelengths of 0.978 A ˚. All diffraction experiments were carried out in a liquid-nitrogen stream at 100 K using a Cryojet XTL system (Oxford Instruments). The diffraction data for DpcA were indexed, integrated and scaled by HKL-3000 [15]. Matthews coefficient was calculated with MATTHEWS_COEF [15], using the the CCP4 software package [16].

Results and discussians

Crystals of DpcA enzyme diffracted to the 1.05 Å resolutions and belonged to P2₁ (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 [16].Structurally DpcA is a member of the superfamily of α/β-hydrolase, molecular replacement with MOLREP [15] from the CCP4 software suite was used for structure solving.

Conclusions

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. DpcA protein has a globular shape and is composed of two domains. The core domain shows composed of eight β-strands, within one is antiparallel (β2). The central β-sheet is flanked on both sides by α-helices: four are on one side and two are on the other side of the sheet. The second domain, the cap structure is located at the C-terminal end of the β-sheet and is composed of α-helices and covers the active site, which will be more reviewed in the presentation. The structure of DpcA is consimilar to the others structurally known HLD.

Acknowledgements

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) and 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). Support of Academy of Sciences of the Czech Republic is appreciated as well.

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