Haloalkane dehalogenases hydrolyze carbon-halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure-function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.
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- bakteriální proteiny chemie genetika metabolismus MeSH
- difrakce rentgenového záření MeSH
- hydrolasy chemie genetika metabolismus MeSH
- krystalizace MeSH
- molekulární sekvence - údaje MeSH
- propan analogy a deriváty chemie metabolismus MeSH
- Rhodococcus enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.
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- 2-propanol MeSH
- bakteriální proteiny chemie MeSH
- difrakce rentgenového záření MeSH
- hydrolasy chemie genetika metabolismus MeSH
- hydrolýza MeSH
- izoenzymy chemie genetika MeSH
- katalýza MeSH
- krystalizace MeSH
- krystalografie rentgenová metody MeSH
- ligandy MeSH
- propan analogy a deriváty MeSH
- Rhodococcus enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
An enzyme-based biosensor was developed by co-immobilization of purified enzyme haloalkane dehalogenase (EC 3.8.1.5) and a fluorescence pH indicator on the tip of an optical fiber. Haloalkane dehalogenase catalyzes hydrolytic dehalogenation of halogenated aliphatic hydrocarbons, which is accompanied by a pH change influencing the fluorescence of the indicator. The pH sensitivity of several fluorescent dyes was evaluated. The selected indicator 5(6)-carboxyfluorescein was conjugated with bovine serum albumin and its reaction was tested under different immobilization conditions. The biosensor was prepared by cross-linking of the conjugate in tandem with haloalkane dehalogenase using glutaraldehyde vapor. The biosensor, stored for 24 h in 50 mM phosphate buffer (pH 7.5) prior to measurement, was used after 15 min of equilibration, the halogenated compound was added, and the response was monitored for 30 min. Calibration of the biosensor with 1,2-dibromoethane and 3-chloro-2-(chloromethyl)-1-propene showed an excellent linear dependence, with detection limits of 0.133 and 0.014 mM, respectively. This biosensor provides a new tool for continuous in situ monitoring of halogenated environmental pollutants.
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- biosenzitivní techniky přístrojové vybavení metody MeSH
- enzymy imobilizované chemie MeSH
- fluorescenční barviva chemie MeSH
- halogenované uhlovodíky analýza chemie MeSH
- molekulární struktura MeSH
- skot MeSH
- technologie optických vláken MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification by the small ubiquitin-like modifier (SUMO) protein plays an important role in mitotic and meiotic recombination. However, the precise role of SUMOylation during recombination is still unclear. Here, we characterize the effect of SUMOylation on the biochemical properties of the Saccharomyces cerevisiae recombination mediator protein Rad52. Interestingly, Rad52 SUMOylation is enhanced by single-stranded DNA, and we show that SUMOylation of Rad52 also inhibits its DNA binding and annealing activities. The biochemical effects of SUMO modification in vitro are accompanied by a shorter duration of spontaneous Rad52 foci in vivo and a shift in spontaneous mitotic recombination from single-strand annealing to gene conversion events in the SUMO-deficient Rad52 mutants. Taken together, our results highlight the importance of Rad52 SUMOylation as part of a 'quality control' mechanism regulating the efficiency of recombination and DNA repair.
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- DNA opravný a rekombinační protein Rad52 chemie metabolismus MeSH
- jednovláknová DNA metabolismus MeSH
- lysin metabolismus MeSH
- oprava DNA MeSH
- protein SUMO-1 metabolismus MeSH
- rekombinace genetická MeSH
- rekombinasa Rad51 metabolismus MeSH
- replikační protein A metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Chirality is a phenomenon that pervades the life on the Earth and has a tremendous importance for our daily lives. Many pharmaceuticals, agrochemicals, food additives, pheromones, fragrances and cosmetics are chiral. Chiral compounds play an essential role in biological systems, mainly for chemical communication among living organisms, serving as sex pheromones, aggregation pheromones, alarm pheromones, trail pheromones, attractants or repellent agents. This review summarizes our current knowledge of the biologically relevant chiral aliphatic hydroxy compounds, which are divided into eight classes according to their chemical structure: primary alcohols, secondary alcohols, tertiary alcohols, glycols and diols, hydroxy ketones, hydroxy carboxylic acids, hydroxy carboxylic esters and hydroxy amines. Information on biological functions and practical applications is summarized for each class. This information could be of interest to chemists, biochemists, biologists and pharmacists. A review with 475 references.
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- biologické přípravky MeSH
- stereoizomerie MeSH
- Publikační typ
- přehledy MeSH
Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.
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.
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- 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
