Crystallographic analysis of new psychrophilic haloalkane dehalogenases: DpcA fromPsychrobacter cryohalolentisK5 and DmxA fromMarinobactersp. ELB17

Abstract

Haloalkane dehalogenases are hydrolytic enzymes with a broad range of potential practical applications such as biodegradation, biosensing, biocatalysis and cellular imaging. Two newly isolated psychrophilic haloalkane dehalogenases exhibiting interesting catalytic properties, DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17, were purified and used for crystallization experiments. After the optimization of crystallization conditions, crystals of diffraction quality were obtained. Diffraction data sets were collected for native enzymes and complexes with selected ligands such as 1-bromohexane and 1,2-dichloroethane to resolutions ranging from 1.05 to 2.49 Å .

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

“…We focusedo ni ns ilico prediction, production,a nd biochemicalc haracterization of five ancestral enzymes correspondingt od ifferent nodes of the HLD-IIp hylogenetic tree and representing the ancestorsofthe thoroughly characterized dehalogenases DbjA, [43,44] DbeA, [45] DhaA, [46] DmxA, [47] and DmmA. [48] The present-daye nzymes display considerable functional variations even thought hey are all closely evolutionary relateda nd share similars tructural topology,t hus providing good modelst oi nvestigate structural andf unctional divergence in the HLD-II subfamily.Characterization of the resurrected ancestral enzymes revealed unique functional properties,including enhanced thermostability,i mproved specific activity,o r modified substrate specificity.T he reconstructed enzymes are the most thermodynamically stable HLDs.…”

“…We focused on in silico prediction, production, and biochemical characterization of five ancestral enzymes corresponding to different nodes of the HLD‐II phylogenetic tree and representing the ancestors of the thoroughly characterized dehalogenases DbjA, DbeA, DhaA, DmxA, and DmmA . The present‐day enzymes display considerable functional variations even though they are all closely evolutionary related and share similar structural topology, thus providing good models to investigate structural and functional divergence in the HLD‐II subfamily.…”

Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17

“…The purified DmxA enzyme in a 50 mM Tris-HCl buffer (pH 7.5) at the concentration of 10 mg/mL was used for the crystallization experiments. The crystallization procedure was performed as described previously by Tratsiak et al [30] with small variation: sitting crystallization drop consisted of 12 µL of the protein solution, 6 µL of precipitant solution, and 3.6 µL of 0.1 M sarcosine (Hampton Research, Aliso Viejo, California, USA) was equilibrated against 2 mL of a reservoir solution in the crystallization mushroom (Triana Science & Technology, Armilla, Spain). The rhombohedral-shaped crystals with the dimensions of approximately 160 µm × 100 µm × 150 µm appeared after 9 days of incubation at 20 °C.…”

Crystal structure of the cold-adapted haloalkane dehalogenase DpcA fromPsychrobacter cryohalolentisK5

“…Crystallization of DpcA and data collection and processing for the crystal in a primitive monoclinic space group have been described previously (Tratsiak et al, 2013). X-ray diffraction data were collected to 1.05 Å resolution on the MX14.2 beamline operated by the Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron-storage ring, Berlin-Adlershof, Germany (Mueller et al, 2015) at a wavelength of 0.978 Å .…”