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 haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications.IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols.
- Klíčová slova
- biotechnology, cosolvents, enzyme, haloalkane dehalogenase, marine, microbial, stability, substrate specificity,
- MeSH
- Bacteria enzymologie genetika metabolismus MeSH
- biokatalýza MeSH
- biotechnologie MeSH
- hydrolasy chemie genetika izolace a purifikace metabolismus MeSH
- katalytická doména MeSH
- katalýza MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- krystalizace MeSH
- metagenom MeSH
- mikrobiální společenstva genetika fyziologie MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
Haloalkane dehalogenases (HLDs) are environmentally relevant enzymes cleaving a carbon-halogen bond in a wide range of halogenated pollutants. PCR with degenerate primers and genome-walking was used for the retrieval of four HLD-encoding genes from groundwater-derived environmental DNA. Using specific primers and the environmental DNA as a template, we succeeded in generating additional amplicons, resulting altogether in three clusters of sequences with each cluster comprising 8-13 closely related putative HLD-encoding genes. A phylogenetic analysis of the translated genes revealed that three HLDs are members of the HLD-I subfamily, whereas one gene encodes an enzyme from the subfamily HLD-II. Two metagenome-derived HLDs, eHLD-B and eHLD-C, each from a different subfamily, were heterologously produced in active form, purified and characterized in terms of their thermostability, pH and temperature optimum, quaternary structure, substrate specificity towards 30 halogenated compounds, and enantioselectivity. eHLD-B and eHLD-C showed striking differences in their activities, substrate preferences, and tolerance to temperature. Profound differences were also determined in the enantiopreference and enantioselectivity of these enzymes towards selected substrates. Comparing our data with those of known HLDs revealed that eHLD-C exhibits a unique combination of high thermostability, high activity, and an unusually broad pH optimum, which covers the entire range of pH 5.5-8.9. Moreover, a so far unreported high thermostability for HLDs was determined for this enzyme at pH values lower than 6.0. Thus, eHLD-C represents an attractive and novel biocatalyst for biotechnological applications.
- Klíčová slova
- Haloalkane dehalogenase, Heterologous production, Metagenomic DNA, Protein stability, Substrate specificity,
- MeSH
- Bacteria genetika MeSH
- bakteriální proteiny genetika izolace a purifikace metabolismus MeSH
- biokatalýza MeSH
- biotechnologie MeSH
- DNA primery MeSH
- fylogeneze MeSH
- hydrolasy genetika izolace a purifikace metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- metagenom * MeSH
- podzemní voda mikrobiologie MeSH
- polymerázová řetězová reakce MeSH
- stabilita proteinů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- DNA primery MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy 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
- Názvy látek
- alkany MeSH
- DNA bakterií MeSH
- estery MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy 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
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 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
Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.
- MeSH
- cirkulární dichroismus MeSH
- denaturace proteinů MeSH
- glykosylace MeSH
- hydrolasy chemie genetika izolace a purifikace metabolismus MeSH
- kinetika MeSH
- klonování DNA MeSH
- konformace proteinů MeSH
- Pichia genetika MeSH
- regulace genové exprese enzymů MeSH
- regulace genové exprese u bakterií MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- Sphingomonas enzymologie genetika MeSH
- stabilita enzymů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
- rekombinantní proteiny 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
To evaluate the potential of using the enzymes from spent mushroom compost (SMC) as an industrial enzyme, the production of alpha-amylase, cellulase, beta-glucosidase, laccase, and xylanase was determined from the SMC of four edible mushroom species (Pleurotus ostreatus, Lentinula edodes, Flammulina velutipes and Hericium erinaceum). Among the tested SMC, the SMC of L. edodes showed the highest enzyme activity in alpha-amylase (229 nkat/g), cellulase (759 nkat/g) and beta-glucosidase (767 nkat/g) in 0.5% Triton X-100, and that of P. ostreatus showed the highest activity in laccase (1452 nkat/g) in phosphate-buffered 0.2% Triton X-100. The highest xylanase activity (119 nkat/g) was found in the SMC of F. velutipes.
- MeSH
- alfa-amylasy izolace a purifikace metabolismus MeSH
- Basidiomycota klasifikace enzymologie metabolismus MeSH
- beta-glukosidasa izolace a purifikace metabolismus MeSH
- biodegradace MeSH
- celulasa izolace a purifikace metabolismus MeSH
- endo-1,4-beta-xylanasy izolace a purifikace metabolismus MeSH
- houby šii-take enzymologie MeSH
- hydrolasy izolace a purifikace metabolismus MeSH
- lakasa izolace a purifikace metabolismus MeSH
- Pleurotus enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- alfa-amylasy MeSH
- beta-glukosidasa MeSH
- celulasa MeSH
- endo-1,4-beta-xylanasy MeSH
- hydrolasy MeSH
- lakasa MeSH
