Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.

• MeSH
• biologické modely MeSH
• hydrolasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• katalytická doména * MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• mutantní proteiny genetika   izolace a purifikace   MeSH
• Mycobacterium avium enzymologie   MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• mutantní proteiny MeSH

Haloalkane dehalogenases degrade halogenated compounds to corresponding alcohols by a hydrolytic mechanism. These enzymes are being intensively investigated as model systems in experimental and in silico studies of enzyme mechanism and evolution, but also hold importance as useful biocatalysts for a number of biotechnological applications. Haloalkane dehalogenases originate from various organisms including bacteria (degraders, symbionts, or pathogens), eukaryotes, and archaea. Several members of this enzyme family have been found in marine organisms. The marine environment represents a good source of enzymes with novel properties, because of its diverse living conditions. A number of novel dehalogenases isolated from marine environments show interesting characteristics such as high activity, unusually broad substrate specificity, stability, or selectivity. In this chapter, the overview of haloalkane dehalogenases from marine organisms is presented and their characteristics are summarized together with an overview of the methods for their identification and biochemical characterization.

• Klíčová slova
• Activity, Biocatalyst, Degradation, Environmental pollutants, Haloalkane dehalogenases, Marine environment, Selectivity, Stability,
• MeSH
• aldehydy metabolismus   MeSH
• alkany metabolismus   MeSH
• biokatalýza MeSH
• biotechnologie metody   MeSH
• enzymatické testy metody   MeSH
• halogeny metabolismus   MeSH
• hydrolasy chemie   izolace a purifikace   metabolismus   MeSH
• substrátová specifita MeSH
• technologie zelené chemie metody   MeSH
• vodní organismy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aldehydy MeSH
• alkany MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• halogeny MeSH
• hydrolasy MeSH

The possibility of integration of the short-end injection mode in the EMMA methodology is demonstrated in this work on the kinetic studies of haloalkane dehalogenase and rhodanese enzymatic reactions. The essential validations of the EMMA methods combined with the short-end and long-end injection modes were performed first to confirm their accuracy. The qualitative and quantitative parameters of both approaches such as repeatabilities of migration times and peak areas, limits of detection and correlation coefficients were in acceptable ranges. In addition, estimated Michaelis constants for the corresponding substrate(s) were comparable being in accordance with previous literature data. Moreover, the ping-pong reaction mechanism of rhodanese reaction was confirmed by means of both injection modes. This combination thus preserves the benefits of these instrumental approaches. Whereas the short-end injection procedure brought 5-6.5 times reduction of the analysis time and 2.5-4 times increase of the sensitivity, the EMMA methodology allowed full automatization of the assays while the whole kinetic studies needed only 20 microl of corresponding enzyme preparation.

• MeSH
• elektroforéza kapilární metody   MeSH
• hydrolasy izolace a purifikace   MeSH
• reprodukovatelnost výsledků MeSH
• substrátová specifita MeSH
• thiosulfátsulfurtransferasa izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• thiosulfátsulfurtransferasa MeSH

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.

• MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• hydrolasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• klonování DNA * MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• Mycobacterium bovis enzymologie   genetika   MeSH
• Mycobacterium klasifikace   enzymologie   genetika   MeSH
• sekvenční analýza DNA MeSH
• skot MeSH
• stabilita enzymů MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata 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 catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.

• MeSH
• alkany metabolismus   MeSH
• fylogeneze MeSH
• genetická transkripce MeSH
• hydrolasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• klonování DNA metody   MeSH
• molekulární sekvence - údaje MeSH
• Mycobacterium avium enzymologie   genetika   růst a vývoj   MeSH
• prasata MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkany MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH