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.
- 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
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.
- 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
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.
- 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
Citrulline ureidase (CTU, EC3.5.1.20) degrades citrulline into ornithine, carbon dioxide, and ammonia. Here, we present the report on expression of recombinant CTU in Escherichia coli. The soluble and active recombinant CTU was expressed in the periplasmic space with the vector pET-22b and the His-tagged CTU was purified with Ni-Affinity Chromatography. The yield of soluble recombinant protein was significantly increased when 1% sorbitol was supplemented in medium. By using phenylisothiocyanate (PITC) pre-column derivatization HPLC, the enzyme activity of recombinant CTU was determined via measuring of the substrate citrulline and the corresponding products. Our results could be useful in the study of CTU biochemical characteristics, enzymatic preparation of ornithine and development of an enzymatic detection method of citrulline.
- MeSH
- Bacteria MeSH
- bakteriologické techniky metody využití MeSH
- chromatografie afinitní metody využití MeSH
- citrulin * izolace a purifikace metabolismus MeSH
- enzymatické testy metody využití MeSH
- Escherichia coli enzymologie imunologie izolace a purifikace MeSH
- Francisella tularensis * enzymologie izolace a purifikace metabolismus MeSH
- hydrolasy izolace a purifikace MeSH
- ornithin * izolace a purifikace metabolismus MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- sorbitol diagnostické užití MeSH
- statistika jako téma MeSH
- tularemie diagnóza etiologie mikrobiologie MeSH
- ureasa izolace a purifikace metabolismus MeSH
- vysokoúčinná kapalinová chromatografie metody využití MeSH
- Publikační typ
- práce podpořená grantem 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
Streptococcus pneumoniae is the main cause of community acquired pneumonia and also produces meningitis, bacteremia, and otitis media, among others. Worldwide, these infections are the cause of substantial morbidity and mortality. Many different virulence factors have been described and most of them are surface-located macromolecules, namely, the capsular polysaccharide and various pneumococcal proteins. Cell wall hydrolases (CWHs) specifically cleave covalent bonds of the peptidoglycan and associated polymers: most CWHs are choline-binding proteins (CBPs) and are among the most well-known surface proteins. Pneumococcal CBPs have been investigated due to their role in pathogenesis and as candidate antigens for improved vaccines. Among the complex host-parasite interactions characteristic of pneumococcal disease, nasopharyngeal colonization is the first step. CBPs appear to play a central role in the development of the carrier state, possibly by affecting biofilm formation and development. Although the role of biofilms in the pathogenesis of some chronic human infections is currently widely accepted, the molecular bases underlying the formation of pneumococcal biofilms are only recently being studied. Among therapeutic strategies to combat multidrug-resistant pneumococcal infections, the use of purified phage- or bacteria-encoded CWHs both in vitro and in animal models is under investigation.
- MeSH
- bakteriální léková rezistence genetika imunologie účinky léků MeSH
- bakteriofágy enzymologie MeSH
- biofilmy růst a vývoj účinky léků MeSH
- buněčná stěna enzymologie účinky léků MeSH
- cholin izolace a purifikace metabolismus MeSH
- enzymy a koenzymy farmakologie terapeutické užití MeSH
- financování organizované MeSH
- hydrolasy izolace a purifikace metabolismus MeSH
- pneumokoková meningitida diagnóza etiologie MeSH
- pneumonie pneumokoková diagnóza etiologie terapie MeSH
- sepse diagnóza etiologie mikrobiologie MeSH
- Streptococcus pneumoniae cytologie izolace a purifikace patogenita MeSH
- Publikační typ
- přehledy 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
- financování organizované MeSH
- glykosylace MeSH
- hydrolasy genetika chemie 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
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 genetika chemie izolace a purifikace metabolismus MeSH
- financování organizované MeSH
- hydrolasy genetika chemie izolace a purifikace metabolismus MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- Mycobacterium bovis enzymologie genetika MeSH
- Mycobacterium enzymologie genetika klasifikace MeSH
- sekvenční analýza DNA MeSH
- skot MeSH
- stabilita enzymů MeSH
- teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- finanční podpora výzkumu jako téma MeSH
- glykosyltransferasy izolace a purifikace metabolismus MeSH
- hydrolasy izolace a purifikace metabolismus MeSH
- isoelektrická fokusace metody MeSH
- izoenzymy izolace a purifikace metabolismus MeSH
- rostliny enzymologie MeSH
