Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.

• Klíčová slova
• Psychrobacter cryohalolentis, X-ray diffraction, haloalkane dehalogenase, psychrophiles, structural analysis, α/β-hydrolase,
• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• genetické vektory chemie   metabolismus   MeSH
• halogenované uhlovodíky chemie   metabolismus   MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• klonování DNA MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• krystalografie rentgenová MeSH
• nízká teplota MeSH
• Psychrobacter chemie   enzymologie   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulového dockingu MeSH
• strukturní homologie proteinů MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 1-bromohexane MeSH Prohlížeč
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• halogenované uhlovodíky MeSH
• hydrolasy MeSH
• rekombinantní fúzní proteiny MeSH

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.

• Klíčová slova
• DmxA, DpcA, Marinobacter sp. ELB17, Psychrobacter cryohalolentis K5, haloalkane dehalogenases,
• MeSH
• bakteriální proteiny analýza   chemie   MeSH
• difrakce rentgenového záření MeSH
• hydrolasy analýza   chemie   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• Marinobacter enzymologie   MeSH
• Psychrobacter enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.

• Klíčová slova
• LinB, Sphingobium japonicum, haloalkane dehalogenase, macroseeding,
• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• biodegradace MeSH
• bromované uhlovodíky chemie   metabolismus   MeSH
• Escherichia coli chemie   genetika   MeSH
• ethylendibromid chemie   metabolismus   MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• izoenzymy chemie   genetika   metabolismus   MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Sphingomonadaceae chemie   enzymologie   genetika   MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bromované uhlovodíky MeSH
• butyl bromide MeSH Prohlížeč
• ethylendibromid MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• izoenzymy MeSH
• rekombinantní proteiny MeSH

A haloalkane dehalogenase, DpcA, from Psychrobacter cryohalolentis K5, representing a novel psychrophilic member of the haloalkane dehalogenase family, was identified and biochemically characterized. DpcA exhibited a unique temperature profile with exceptionally high activities at low temperatures. The psychrophilic properties of DpcA make this enzyme promising for various environmental applications.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• fyziologická adaptace * MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• nízká teplota * MeSH
• Psychrobacter enzymologie   genetika   růst a vývoj   fyziologie   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

• MeSH
• 2-propanol MeSH
• bakteriální proteiny chemie   MeSH
• difrakce rentgenového záření MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• hydrolýza MeSH
• izoenzymy chemie   genetika   MeSH
• katalýza MeSH
• krystalizace MeSH
• krystalografie rentgenová metody   MeSH
• ligandy MeSH
• propan analogy a deriváty   MeSH
• Rhodococcus enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• 1,2,3-trichloropropane MeSH Prohlížeč
• 2-propanol MeSH
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• izoenzymy MeSH
• ligandy MeSH
• propan MeSH