Chovancova E

References (4)

Title : A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB - Biedermannova_2012_J.Biol.Chem_287_29062
Author(s) : Biedermannova L , Prokop Z , Gora A , Chovancova E , Kovacs M , Damborsky J , Wade RC
Ref : Journal of Biological Chemistry , 287 :29062 , 2012
Abstract : Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.
ESTHER : Biedermannova_2012_J.Biol.Chem_287_29062
PubMedSearch : Biedermannova_2012_J.Biol.Chem_287_29062
PubMedID: 22745119

Title : Biochemical characterization of a novel haloalkane dehalogenase from a cold-adapted bacterium - Drienovska_2012_Appl.Environ.Microbiol_78_4995
Author(s) : Drienovska I , Chovancova E , Koudelakova T , Damborsky J , Chaloupkova R
Ref : Applied Environmental Microbiology , 78 :4995 , 2012
Abstract : 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.
ESTHER : Drienovska_2012_Appl.Environ.Microbiol_78_4995
PubMedSearch : Drienovska_2012_Appl.Environ.Microbiol_78_4995
PubMedID: 22582053
Gene_locus related to this paper: psyck-q1qbb9

Title : Substrate specificity of haloalkane dehalogenases - Koudelakova_2011_Biochem.J_435_345
Author(s) : Koudelakova T , Chovancova E , Brezovsky J , Monincova M , Fortova A , Jarkovsky J , Damborsky J
Ref : Biochemical Journal , 435 :345 , 2011
Abstract : 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.
ESTHER : Koudelakova_2011_Biochem.J_435_345
PubMedSearch : Koudelakova_2011_Biochem.J_435_345
PubMedID: 21294712
Gene_locus related to this paper: agrtu-DHAA , brael-e2rv62 , braja-dhaa , myctu-linb , myctu-Rv1833c , rhoba-DHLA , rhoso-halo1 , sphpi-linb , xanau-halo1

Title : Phylogenetic analysis of haloalkane dehalogenases - Chovancova_2007_Proteins_67_305
Author(s) : Chovancova E , Kosinski J , Bujnicki JM , Damborsky J
Ref : Proteins , 67 :305 , 2007
Abstract : Haloalkane dehalogenases (HLDs) are enzymes that catalyze the cleavage of carbon-halogen bonds by a hydrolytic mechanism. Although comparative biochemical analyses have been published, no classification system has been proposed for HLDs, to date, that reconciles their phylogenetic and functional relationships. In the study presented here, we have analyzed all sequences and structures of genuine HLDs and their homologs detectable by database searches. Phylogenetic analyses revealed that the HLD family can be divided into three subfamilies denoted HLD-I, HLD-II, and HLD-III, of which HLD-I and HLD-III are predicted to be sister-groups. A mismatch between the HLD protein tree and the tree of species, as well as the presence of more than one HLD gene in a few genomes, suggest that horizontal gene transfers, and perhaps also multiple gene duplications and losses have been involved in the evolution of this family. Most of the biochemically characterized HLDs are found in the HLD-II subfamily. The dehalogenating activity of two members of the newly identified HLD-III subfamily has only recently been confirmed, in a study motivated by this phylogenetic analysis. A novel type of the catalytic pentad (Asp-His-Asp+Asn-Trp) was predicted for members of the HLD-III subfamily. Calculation of the evolutionary rates and lineage-specific innovations revealed a common conserved core as well as a set of residues that characterizes each HLD subfamily. The N-terminal part of the cap domain is one of the most variable regions within the whole family as well as within individual subfamilies, and serves as a preferential site for the location of relatively long insertions. The highest variability of discrete sites was observed among residues that are structural components of the access channels. Mutations at these sites modify the anatomy of the channels, which are important for the exchange of ligands between the buried active site and the bulk solvent, thus creating a structural basis for the molecular evolution of new substrate specificities. Our analysis sheds light on the evolutionary history of HLDs and provides a structural framework for designing enzymes with new specificities.
ESTHER : Chovancova_2007_Proteins_67_305
PubMedSearch : Chovancova_2007_Proteins_67_305
PubMedID: 17295320