Structural comparison of DpcA and DmxA, α/β-hydrolase fold family members from different HLD subfamilies

Katsiaryna Tratsiak1, Lukáš Chrást2, Tatyana Prudnikova1, Ivana Drienovská2,
Jiří Damborský2, Oksana Degtjarik1, Pavlína Řezáčová3,4, Michal Kutý1,5,
Radka Chaloupková2, Ivana Kutá Smatanová1,5

1 University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 31, 370 05 České Budějovice, Czech Republic, *E-mail: ktratsiak@gmail.com
2 Masaryk University, Faculty of Science, Brno, Czech Republic
3 Academy 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 enzyme DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17, catalyzing the hydrolytic conversion of halogenated aliphatic compounds with  releasing a corresponding alcohol, a halide ion, and a proton as the reaction products belong to the haloalkane dehalogenases (EC 3.8.1.5; HLD), HLD-I and HLD-II subfamilies, respectfully. The enzymes are potentially practical 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.

DpcA and DmxA own unique temperature profiles with exceptionally high activities at low temperatures for DpcA (25 °C, pH 8.7, towards 1,3-dibromopropane)  and height temperatures for DmxA (the maximal activity towards 1,3-diiodopropane was detected at 55 °C, pH 9.0), what highlights them among the other HLDs.

The crystallized crystals of DpcA diffracted to the resolution 1.05 Å, beamline 14.2 (BESSY II electron-storage ring, HZB, Germany), P21 space group and DmxA to the resolution 1.45 Å, beamline ID29, at the (ESRF, Grenoble, France), P212121 space group.

The structure were solved by molecular replacement with MOLREP from the CCP4 software suite by using the coordinates of 1B6G (40% sequence identities and 53% sequence similarity) was used as search model for DpcA structure and 4E46 for DmxA (48% sequence identity and 63% sequence similarity).

The proteins have a globular shape and are composed of two domains: a highly conserved main domain, which 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 the activity of DpcA: Asp-123, His-280,  Asp- 250, Trp-124,Trp-164 and for  DmxA: Asp 105, His 273, Glu 129, Gln 40 , Trp106.

DpcA has one molecule in assymetric unit and DmxA – two, 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.