3Institute of Organic Chemistry and Biochemistry, ASCR, Flemingovo namesti 2, Prague, Czech Republic
4Institute of Microbiology, Center for Nanobiology and Structural Biology, ASCR, Zamek 136, Nove Hrady, Czech Republic
5Institute of Biochemistry, University of Lubeck, Ratzeburger Allee 160, Lubeck, Germany
6Biological Sciences, University of Alabama in Huntsville, 301 Sparkman Dr., Huntsville, AL, USA
The HAD (haloacid dehalogenase) superfamily is one of the largest known group of enzymes, the majority of them catalyze the hydrolysis of phosphoric acid monoesters into a phosphate ion and an alcohol. Despite the fact that sequence similarity between HAD phosphatases is generally very low, the members possess some characteristic features, such as Rossmann-like fold, HAD signature motifs or the requirement for Mg2+ ion as an obligatory cofactor. This study was focused on new hypothetical HAD phosphatase from Thermococcus thioreducens, that can possibly degradate phosphorylated compounds. The protein crystallized in space group P21212 with unit-cell parameters a = 66.3, b = 117.0, c = 33.8 Å, and the crystals contained one molecule in the asymmetric unit. The protein structure was determined by X-ray crystallography and refined to 1.75 Å resolution. The structure revealed a putative active site, common to all HAD members. Computational docking into the crystal structure was used to propose substrates for the enzyme. Activity of this thermophilic enzyme towards selected substrates was confirmed at temperature 333 K.
We would particularly like to acknowledge the help and support of Dr. Manfred Weiss and Dr. Christian Feiler during the diffraction experiment.
The work was supported by GACR 17-24321S, GAJU 04-149/2016/P, DAAD-16-09, ERDF CZ.02.1.01/0.0/0.0/15_003/0000441, RVO 68378050 and RVO 61388963.