Silymarin is known for its hepatoprotective effects. Although there is solid evidence for its protective effects against Amanita phalloides intoxication, only inconclusive data are available for alcoholic liver damage. Since silymarin flavonolignans have metal-chelating activity, we hypothesized that silymarin may influence alcoholic liver damage by inhibiting zinc-containing alcohol dehydrogenase (ADH). Therefore, we tested the zinc-chelating activity of pure silymarin flavonolignans and their effect on yeast and equine ADH. The most active compounds were also tested on bovine glutamate dehydrogenase, an enzyme blocked by zinc ions. Of the six flavonolignans tested, only 2,3-dehydroderivatives (2,3-dehydrosilybin and 2,3-dehydrosilychristin) significantly chelated zinc ions. Their effect on yeast ADH was modest but stronger than that of the clinically used ADH inhibitor fomepizole. In contrast, fomepizole strongly blocked mammalian (equine) ADH. 2,3-Dehydrosilybin at low micromolar concentrations also partially inhibited this enzyme. These results were confirmed by in silico docking of active dehydroflavonolignans with equine ADH. Glutamate dehydrogenase activity was decreased by zinc ions in a concentration-dependent manner, and this inhibition was abolished by a standard zinc chelating agent. In contrast, 2,3-dehydroflavonolignans blocked the enzyme both in the absence and presence of zinc ions. Therefore, 2,3-dehydrosilybin might have a biologically relevant inhibitory effect on ADH and glutamate dehydrogenase.
- MeSH
- alkoholdehydrogenasa antagonisté a inhibitory MeSH
- chelátory farmakologie MeSH
- flavonolignany farmakologie MeSH
- glutamátdehydrogenasa antagonisté a inhibitory MeSH
- koně MeSH
- kvasinky účinky léků MeSH
- silibinin farmakologie MeSH
- silymarin farmakologie MeSH
- zinek izolace a purifikace metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free -OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN3). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin).
- MeSH
- Aspergillus niger enzymologie MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny chemie metabolismus MeSH
- glykosidhydrolasy chemie metabolismus MeSH
- glykosylace MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- rekombinantní proteiny metabolismus MeSH
- rutin chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
Photosystem II (PSII) is a multi-subunit pigment-protein complex and is one of several protein assemblies that function cooperatively in photosynthesis in plants and cyanobacteria. As more structural data on PSII become available, new questions arise concerning the nature of the charge separation in PSII reaction center (RC). The crystal structure of PSII RC from cyanobacteria Thermosynechococcus vulcanus was selected for the computational study of conformational changes in photosystem II associated to the charge separation process. The parameterization of cofactors and lipids for classical MD simulation with Amber force field was performed. The parametrized complex of PSII was embedded in the lipid membrane for MD simulation with Amber in Gromacs. The conformational behavior of protein and the cofactors directly involved in the charge separation were studied by MD simulations and QM/MM calculations. This study identified the most likely mechanism of the proton-coupled reduction of plastoquinone QB. After the charge separation and the first electron transfer to QB, the system undergoes conformational change allowing the first proton transfer to QB- mediated via Ser264. After the second electron transfer to QBH, the system again adopts conformation allowing the second proton transfer to QBH-. The reduced QBH2 would then leave the binding pocket.
The haloacid dehalogenase (HAD) superfamily is one of the largest known groups of enzymes and the majority of its members catalyze the hydrolysis of phosphoric acid monoesters into a phosphate ion and an alcohol. Despite the fact that sequence similarity between HAD phosphatases is generally very low, the members of the family possess some characteristic features, such as a Rossmann-like fold, HAD signature motifs or the requirement for Mg2+ ion as an obligatory cofactor. This study focuses on a new hypothetical HAD phosphatase from Thermococcus thioreducens. The protein crystallized in space group P21212, with unit-cell parameters a = 66.3, b = 117.0, c = 33.8 Å, and the crystals contained one molecule in the asymmetric unit. The protein structure was determined by X-ray crystallography and was refined to 1.75 Å resolution. The structure revealed a putative active site common to all HAD members. Computational docking into the crystal structure was used to propose substrates of the enzyme. The activity of this thermophilic enzyme towards several of the selected substrates was confirmed at temperatures of 37°C as well as 60°C.
Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- genetické vektory chemie metabolismus MeSH
- halogenované uhlovodíky chemie metabolismus MeSH
- hydrolasy chemie genetika metabolismus MeSH
- interakční proteinové domény a motivy MeSH
- klonování DNA MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- krystalografie rentgenová MeSH
- nízká teplota MeSH
- Psychrobacter chemie enzymologie MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- simulace molekulového dockingu MeSH
- strukturní homologie proteinů MeSH
- substrátová specifita MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- biodegradace MeSH
- hydrolasy chemie metabolismus MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- látky znečišťující životní prostředí chemie metabolismus MeSH
- molekulární modely MeSH
- mutageneze MeSH
- propan analogy a deriváty chemie metabolismus MeSH
- Rhodococcus chemie enzymologie MeSH
- sekundární struktura proteinů MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- biodegradace MeSH
- bromované uhlovodíky chemie metabolismus MeSH
- Escherichia coli chemie genetika MeSH
- ethylendibromid chemie metabolismus MeSH
- hydrolasy chemie genetika metabolismus MeSH
- izoenzymy chemie genetika metabolismus MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- Sphingomonadaceae chemie enzymologie genetika MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.
- MeSH
- Arabidopsis metabolismus MeSH
- difrakce rentgenového záření MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fosfotransferasy chemie izolace a purifikace MeSH
- krystalizace MeSH
- proteiny huseníčku chemie izolace a purifikace MeSH
- signální transdukce * MeSH
- tranzitní teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Å resolution and are not analysed further here. The 1.2 Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.
- MeSH
- difrakce rentgenového záření MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- flavinmononukleotid chemie metabolismus MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- NAD(P)H dehydrogenasa (chinon) chemie metabolismus MeSH
- oxidace-redukce MeSH
- proteiny z Escherichia coli chemie metabolismus MeSH
- represorové proteiny chemie metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
