Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 Å resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.

• Klíčová slova
• access tunnel, catalytic pentad, dimer, enantiselectivity, haloalkane dehalogenase, psychrophile, thermostability,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• active site, conformational change, dichloroethane degradation, enzyme catalysis, enzyme kinetics, enzyme mechanism, ethylene dichloride, haloalkane dehalogenase, molecular dynamics, molecular evolution, organic halogen, organohalogen, protein conformation,
• 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
• Názvy látek
• ethylendichloridy MeSH
• ethylene dichloride MeSH Prohlížeč
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• kumariny MeSH

Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

• MeSH
• algoritmy * MeSH
• hydrolasy chemie   metabolismus   MeSH
• konformace proteinů * MeSH
• krystalografie MeSH
• proteiny chemie   metabolismus   MeSH
• shluková analýza MeSH
• simulace molekulární dynamiky MeSH
• software * MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• proteiny MeSH

Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.

• MeSH
• biodegradace MeSH
• cirkulární dichroismus MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• látky znečišťující životní prostředí chemie   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• počítačová simulace MeSH
• propan analogy a deriváty   chemie   MeSH
• proteinové inženýrství * MeSH
• Rhodococcus enzymologie   genetika   růst a vývoj   MeSH
• řízená evoluce molekul MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2,3-trichloropropane MeSH Prohlížeč
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• látky znečišťující životní prostředí MeSH
• propan MeSH

This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.

• MeSH
• Bacteria enzymologie   MeSH
• cirkulární dichroismus MeSH
• DNA bakterií chemie   genetika   MeSH
• hydrolasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• Mycobacterium bovis enzymologie   MeSH
• sekvenční analýza DNA MeSH
• substrátová specifita MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy 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
• 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
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH
• rozpouštědla MeSH

1,2,3-Trichloropropane (TCP) is a highly toxic, recalcitrant byproduct of epichlorohydrin manufacture. Haloalkane dehalogenase (DhaA) from Rhodococcus sp. hydrolyses the carbon-halogen bond in various halogenated compounds including TCP, but with low efficiency (k (cat)/K (m )= 36 s(-1) M(-1)). A Cys176Tyr-DhaA mutant with a threefold higher catalytic efficiency for TCP dehalogenation has been previously obtained by error-prone PCR. We have used molecular simulations and quantum mechanical calculations to elucidate the molecular mechanisms involved in the improved catalysis of the mutant, and enantioselectivity of DhaA toward TCP. The Cys176Tyr mutation modifies the protein access and export routes. Substitution of the Cys residue by the bulkier Tyr narrows the upper tunnel, making the second tunnel "slot" the preferred route. TCP can adopt two major orientations in the DhaA enzyme, in one of which the halide-stabilizing residue Asn41 forms a hydrogen bond with the terminal halogen atom of the TCP molecule, while in the other it bonds with the central halogen atom. The differences in these binding patterns explain the preferential formation of the (R)- over the (S)-enantiomer of 2,3-dichloropropane-1-ol in the reaction catalyzed by the enzyme.

• MeSH
• hydrolasy genetika   metabolismus   MeSH
• molekulární modely * MeSH
• mutace MeSH
• polymerázová řetězová reakce MeSH
• propan analogy a deriváty   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2,3-trichloropropane MeSH Prohlížeč
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• propan MeSH