Structure-functional analysis of the haloalkane dehalogenase DmxA from Marinobacter sp. ELB17

Katsiaryna Tratsiak^{* 1}, Tatyana Prudnikova ¹,  Lukas Chrast ², Jiri Damborsky ², Oksana Degtjarik ¹, Pavlina Rezacova ^3,4,  Radka Chaloupkova ², Michal Kuty ^1,5and 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

²Masaryk University, Faculty of Science, Brno, Czech Republic

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

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

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

The selected enzyme DmxA, 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, was isolated from Marinobacter sp. ELB 17 (DmxA). DmxA is one of the 10 members of α/β-hydrolases, which are already on the market for the practical use for such applications as biodegradation, biosensing, protein tagging for cell imaging and protein analysis, decontamination of warfare agents, production of optically active hydrocarbons and alcohols.

DmxA is an exctremoenzyme, exhibiting height enantioselectivity, however reveals the highest activity at height temperatures (the maximal activity towards 1,3-diiodopropane was detected at 55 °C and pH 9.0) , what highlights it among the other HLDs.

Diffracted crystals of DmxA were refined up to the resolutions 1.45 Å. Diffraction data for DmxA were collected using Pilatus 6M-F detector at the wavelengths of 0.972 Å on the beamline ID29, at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France).

Crystal of DmxA belonged to P2₁2₁2₁ space group, with the 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 structure was solved by molecular replacement with MOLREP from the CCP4 software suite by using the coordinates of Rhodococcus rhodochrous (PDB entry 4E46; 48% sequence identity for 142 residues and 63% sequence similarity).

Structurally DmxA showed the typical composition of the molecule of the other members of the superfamily of α/β - hydrolases. The proteins have a globular shape and are composed of two domains: a highly conserved α/β - hydrolase main domain, which is the scaffold - like for the catalytic residues, and a smaller helical cap domain. The core domain is composed of eight β - strands, within 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 β - sheet. The second domain, the cap structure is located at the C - terminal end of the β - sheet and is composed of α - helices, covering the active site, which has revealed the catalytic pentad essential for their activity: Asp 105, His 273, Glu 129, Gln 40 , Trp106. Presenting on the both in monomeric and dimeric form, a dimer enzyme of the  a uniquely formed by the covalent disulfide through Cys 294, the homo- dimer is chosen as biological asymmetric unit.

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.