- Publikační typ
- abstrakt z konference MeSH
We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.
- MeSH
- bromované uhlovodíky chemie MeSH
- fluorescenční spektrometrie MeSH
- hydrolasy chemie genetika MeSH
- katalytická doména MeSH
- katalýza MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená MeSH
- proteinové inženýrství * MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- stereoizomerie MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β-hydrolases which act to cleave carbon-halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high robustness, the members of this enzyme family have been employed in several practical applications: (i) biocatalytic preparation of optically pure building-blocks for organic synthesis; (ii) recycling of by-products from chemical processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein analysis. This review discusses the application of HLDs in the context of the biochemical properties of individual enzymes. Further extension of HLD uses within the field of biotechnology will require currently limiting factors - such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of organic co-solvents - to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from extremophilic microorganisms may offer solutions.
Haloalkane dehalogenases are known as bacterial enzymes cleaving a carbon-halogen bond in halogenated compounds. Here we report the first biochemically characterized non-microbial haloalkane dehalogenase DspA from Strongylocentrotus purpuratus. The enzyme shows a preference for terminally brominated hydrocarbons and enantioselectivity towards β-brominated alkanes. Moreover, we identified other putative haloalkane dehalogenases of eukaryotic origin, representing targets for future experiments to discover dehalogenases with novel catalytic properties.
- MeSH
- cirkulární dichroismus MeSH
- halogeny chemie MeSH
- hydrolasy chemie genetika MeSH
- katalýza MeSH
- klonování DNA MeSH
- regulace genové exprese enzymů MeSH
- sekvence aminokyselin genetika MeSH
- Strongylocentrotus purpuratus enzymologie genetika MeSH
- substrátová specifita MeSH
- uhlík chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.
- MeSH
- Agrobacterium tumefaciens enzymologie genetika metabolismus MeSH
- alkany metabolismus MeSH
- DNA bakterií chemie genetika MeSH
- estery metabolismus MeSH
- hydrolasy genetika izolace a purifikace metabolismus MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- rostliny mikrobiologie MeSH
- sekvenční analýza DNA MeSH
- stereoizomerie MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.
- MeSH
- Agrobacterium tumefaciens enzymologie genetika metabolismus MeSH
- aktivace enzymů MeSH
- biologické modely MeSH
- Bradyrhizobium enzymologie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- fylogeneze MeSH
- hydrolasy klasifikace genetika metabolismus fyziologie MeSH
- mutantní proteiny klasifikace genetika metabolismus MeSH
- Mycobacterium bovis enzymologie genetika metabolismus MeSH
- Mycobacterium smegmatis genetika metabolismus MeSH
- Rhodococcus enzymologie genetika metabolismus MeSH
- Sphingobacterium enzymologie genetika metabolismus MeSH
- substrátová specifita MeSH
- Xanthobacter enzymologie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 A, respectively.
