Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.
- MeSH
- ethylendichloridy metabolismus MeSH
- halogenace MeSH
- hydrolasy chemie metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kumariny chemie metabolismus MeSH
- metylace MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Xanthobacter chemie enzymologie metabolismus 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
BACKGROUND: The main aim of this study was to develop and implement an algorithm for the rapid, accurate and automated identification of paths leading from buried protein clefts, pockets and cavities in dynamic and static protein structures to the outside solvent. RESULTS: The algorithm to perform a skeleton search was based on a reciprocal distance function grid that was developed and implemented for the CAVER program. The program identifies and visualizes routes from the interior of the protein to the bulk solvent. CAVER was primarily developed for proteins, but the algorithm is sufficiently robust to allow the analysis of any molecular system, including nucleic acids or inorganic material. Calculations can be performed using discrete structures from crystallographic analysis and NMR experiments as well as with trajectories from molecular dynamics simulations. The fully functional program is available as a stand-alone version and as plug-in for the molecular modeling program PyMol. Additionally, selected functions are accessible in an online version. CONCLUSION: The algorithm developed automatically finds the path from a starting point located within the interior of a protein. The algorithm is sufficiently rapid and robust to enable routine analysis of molecular dynamics trajectories containing thousands of snapshots. The algorithm is based on reciprocal metrics and provides an easy method to find a centerline, i.e. the spine, of complicated objects such as a protein tunnel. It can also be applied to many other molecules. CAVER is freely available from the web site http://loschmidt.chemi.muni.cz/caver/.
- MeSH
- algoritmy MeSH
- financování organizované MeSH
- internet MeSH
- konformace proteinů MeSH
- krystalografie rentgenová metody MeSH
- magnetická rezonanční spektroskopie MeSH
- počítačová simulace MeSH
- proteiny chemie MeSH
- proteomika metody MeSH
- Rhodococcus MeSH
- rozpouštědla chemie MeSH
- sekundární struktura proteinů MeSH
- software MeSH
- Sphingomonas MeSH
- Xanthobacter MeSH
