Iripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3 Å and the structure of the cleaved conformation was solved at 2.0 Å resolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.

• Klíčová slova
• Iripin-4, Ixodes ricinus, X-ray structure, cleaved conformation, native conformation, serpins,
• MeSH
• arginin MeSH
• klíště * MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• serpiny * chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• serpiny * MeSH

The current chelation therapy has several drawbacks, including lack of selectivity, which could lead to trace metal depletion. Consequently, the proper function of metalloenzymes can be disrupted. Flavonoids possess chelating properties and hence interfere with the homeostasis of essential metals. We focused on zinc, an important trace metal required for the function of many enzymes and transcription factors. After making an initial evaluation of the Zn2+-chelating properties of a series of flavonoids, the effect of these compounds on various zinc-containing enzymes was also investigated. We performed enzyme inhibition assays spectrophotometrically using yeast and equine alcohol dehydrogenases and bovine glutamate dehydrogenase. Nine of the 21 flavonoids tested were capable of chelating Zn2+. Baicalein and 3-hydroxyflavone were the most potent Zn2+ chelators under slightly acidic and neutral pH conditions. This chelation was also confirmed by the ability to reverse Zn2+-induced enzymatic inhibition of bovine glutamate dehydrogenase. Although some flavonoids were also able to inhibit zinc-containing alcohol dehydrogenases, this inhibition was likely not caused by Zn2+ chelation. Luteolin was a relatively potent inhibitor of these enzymes regardless of the presence of Zn2+. Docking studies confirmed the binding of active flavonoids to equine alcohol dehydrogenase without any significant interaction with the catalytic zinc.

• Klíčová slova
• alcohol dehydrogenase, chelation, enzymatic, glutamate dehydrogenase, inhibition, zinc,
• MeSH
• alkoholdehydrogenasa metabolismus   MeSH
• chelátory chemie   MeSH
• flavonoidy * MeSH
• glutamátdehydrogenasa MeSH
• koně MeSH
• kovy metabolismus   MeSH
• skot MeSH
• zinek * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alkoholdehydrogenasa MeSH
• chelátory MeSH
• flavonoidy * MeSH
• glutamátdehydrogenasa MeSH
• kovy MeSH
• zinek * MeSH

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.

• Klíčová slova
• alcohol dehydrogenase, chelation, dehydrosilybin, docking, flavonolignans, glutamate dehydrogenase, silybin, zinc,
• 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
• Názvy látek
• alkoholdehydrogenasa MeSH
• chelátory MeSH
• flavonolignany MeSH
• glutamátdehydrogenasa MeSH
• silibinin MeSH
• silymarin MeSH
• zinek MeSH

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

• Klíčová slova
• ancestral sequence reconstruction, haloalkane dehalogenase, halogenated pollutants, structural analysis,
• MeSH
• hydrolasy chemie   metabolismus   MeSH
• hydrolýza MeSH
• katalytická doména MeSH
• krystalografie rentgenová metody   MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• Mycobacterium bovis enzymologie   MeSH
• proteinové inženýrství metody   MeSH
• sekvenční analýza proteinů metody   MeSH
• Sphingomonadaceae enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

• Klíčová slova
• Iripin-5, Ixodes ricinus, X-ray structure, serine protease inhibitors, serpins, tick saliva,
• MeSH
• antiflogistika * chemie   izolace a purifikace   MeSH
• erytrocyty MeSH
• inhibitory enzymů * chemie   izolace a purifikace   MeSH
• klíště metabolismus   MeSH
• králíci MeSH
• kultivované buňky MeSH
• makrofágy MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neutrofily MeSH
• serpiny * chemie   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiflogistika * MeSH
• inhibitory enzymů * MeSH
• serpiny * MeSH

Haloalkane dehalogenases (EC 3.8.1.5) are microbial enzymes that catalyse the hydrolytic conversion of halogenated compounds, resulting in a halide ion, a proton and an alcohol. These enzymes are used in industrial biocatalysis, bioremediation and biosensing of environmental pollutants or for molecular tagging in cell biology. The novel haloalkane dehalogenase DpaA described here was isolated from the psychrophilic and halophilic bacterium Paraglaciecola agarilytica NO2, which was found in marine sediment collected from the East Sea near Korea. Gel-filtration experiments and size-exclusion chromatography provided information about the dimeric composition of the enzyme in solution. The DpaA enzyme was crystallized using the sitting-drop vapour-diffusion method, yielding rod-like crystals that diffracted X-rays to 2.0 Å resolution. Diffraction data analysis revealed a case of merohedral twinning, and subsequent structure modelling and refinement resulted in a tetrameric model of DpaA, highlighting an uncommon multimeric nature for a protein belonging to haloalkane dehalogenase subfamily I.

• Klíčová slova
• DpaA, crystallization, haloalkane dehalogenases, halogenated pollutants, oligomerization, tetrameric structure,
• MeSH
• Alteromonadaceae enzymologie   MeSH
• bakteriální proteiny chemie   MeSH
• biodegradace MeSH
• hydrofobní a hydrofilní interakce MeSH
• hydrolasy chemie   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• sekvenční seřazení MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy 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).

• Klíčová slova
• azide, glycosylation, molecular modeling, rutinosidase, tunnel,
• 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
• Názvy látek
• disacharidy MeSH
• fungální proteiny MeSH
• glykosidhydrolasy MeSH
• rekombinantní proteiny MeSH
• rutin MeSH
• rutinose MeSH Prohlížeč

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.

• Klíčová slova
• MD simulations, Photosystem II reaction center, Plastoquinone, Proton-coupled reduction,
• MeSH
• bakteriální proteiny chemie   MeSH
• fotosystém II (proteinový komplex) chemie   MeSH
• lipidové dvojvrstvy chemie   MeSH
• simulace molekulární dynamiky * MeSH
• sinice enzymologie   MeSH
• Thermosynechococcus MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fotosystém II (proteinový komplex) MeSH
• lipidové dvojvrstvy MeSH

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.

• Klíčová slova
• HAD superfamily, crystal structure, docking, hypothetical phosphatase, phosphatase assay,
• MeSH
• fosfatasy chemie   MeSH
• hydrolasy chemie   MeSH
• katalytická doména MeSH
• kinetika MeSH
• krystalografie rentgenová metody   MeSH
• molekulární modely MeSH
• substrátová specifita MeSH
• Thermococcus enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2-haloacid dehalogenase MeSH Prohlížeč
• fosfatasy MeSH
• hydrolasy MeSH

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.

• Klíčová slova
• Psychrobacter cryohalolentis, X-ray diffraction, haloalkane dehalogenase, psychrophiles, structural analysis, α/β-hydrolase,
• 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
• Názvy látek
• 1-bromohexane MeSH Prohlížeč
• bakteriální proteiny MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• halogenované uhlovodíky MeSH
• hydrolasy MeSH
• rekombinantní fúzní proteiny MeSH