In the last decades, the structural flexibility of cytochromes P450 has been extensively studied by spectroscopic and in silico methods. Here, both approaches are reviewed and compared. Comparison of both methods indicates that the individual cytochromes P450 differ significantly in the flexibilities of their substrate-binding active sites. This finding probably accounts for the large number of isoforms of these enzymes (there are fifty-seven known cytochrome P450 genes in the human genome) and their functional versatility. On the other hand, most of the known cytochrome P450s have a set of common structural features, with an overall structure consisting of a relatively flexible domain (the distal side), a more rigid domain (the heme-binding core) and a domain on the proximal side of the hemoprotein with intermediate flexibility. Substrate access and product egress channels of CYP enzymes are also important structural elements as the majority of these channels are located in the flexible distal side; the location, flexibility, and function of these channels are discussed.

• MeSH
• konformace proteinů MeSH
• lidé MeSH
• simulace molekulární dynamiky MeSH
• spektrální analýza metody   MeSH
• substrátová specifita MeSH
• systém (enzymů) cytochromů P-450 chemie   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Molecular dynamics (MD) simulations at normal and high temperature were used to study the flexibility and malleability of three microsomal cytochromes P450 (CYPs): CYP3A4, CYP2C9, and CYP2A6. Comparison of B-factors (describing the atomic fluctuations) between X-ray and MD data shows that the X-ray B-factors are significantly lower in the regions where the crystal contacts occur than for other regions. Consequently, the conclusions about CYP flexibility based solely on the X-ray data might be misleading. Comparison of flexibility patterns of the three CYPs enabled common features and variations in flexibility and malleability of the studied CYPs to be identified. The previously described pattern of flexibility in topological elements of microsomal CYPs (a rigid heme binding core, a malleable distal side and intermediately flexible proximal side) was confirmed. These topological features provide an important combination of high stereo- and regio-specificity (mediated by the relative rigidity in the neighborhood of the heme), together with high substrate promiscuity due to the more flexible active site and the malleability of the distal side. The data acquired here show that the malleability of the three studied CYPs correlates with their substrate specificity: CYP2A6 has a narrow substrate range and is the most rigid, CYP3A4 is the most promiscuous CYP known and is the most malleable, and CYP2C9 is intermediate in terms of both its substrate specificity and malleability. Thus, the malleability of CYPs is probably a major determinant of their substrate specificity.

• MeSH
• aromatické hydroxylasy chemie   metabolismus   MeSH
• cytochrom P-450 CYP3A chemie   metabolismus   MeSH
• financování organizované MeSH
• játra metabolismus   MeSH
• konformace proteinů MeSH
• kumariny metabolismus   MeSH
• léčivé přípravky metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• nikotin metabolismus   MeSH
• steroidy metabolismus   MeSH
• substrátová specifita MeSH
• systém (enzymů) cytochromů P-450 chemie   metabolismus   MeSH
• tabák metabolismus   MeSH
• teplota MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH

BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

• MeSH
• beta-N-acetylhexosaminidasy chemie   metabolismus   MeSH
• fylogeneze MeSH
• glykosylace MeSH
• katalytická doména MeSH
• kinetika MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• reprodukovatelnost výsledků MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• Talaromyces enzymologie   MeSH
• výpočetní biologie * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A flexible transceiver array based on transmission line resonators (TLRs) combining the advantages of coil arrays with the possibility of form-fitting targeting cardiac MRI at 7 T is presented. The design contains 12 elements which are fabricated on a flexible substrate with rigid PCBs attached on the center of each element to place the interface components, i.e. transmit/receive (T/R) switch, power splitter, pre-amplifier and capacitive tuning/matching circuitry. The mutual coupling between elements is cancelled using a decoupling ring-based technique. The performance of the developed array is evaluated by 3D electromagnetic simulations, bench tests, and MR measurements using phantoms. Efficient inter-element decoupling is demonstrated in flat configuration on a box-shaped phantom (Sij < -19 dB), and bent on a human torso phantom (Sij < -16 dB). Acceleration factors up to 3 can be employed in bent configuration with reasonable g-factors (<1.7) in an ROI at the position of the heart. The array enables geometrical conformity to bodies within a large range of size and shape and is compatible with parallel imaging and parallel transmission techniques.

• MeSH
• elektromagnetická pole MeSH
• fantomy radiodiagnostické MeSH
• lidé MeSH
• magnetická rezonanční tomografie přístrojové vybavení   MeSH
• počítačová simulace MeSH
• poměr signál - šum MeSH
• rádiové vlny MeSH
• srdce diagnostické zobrazování   MeSH
• trup diagnostické zobrazování   MeSH
• vylepšení obrazu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We analyze the conditions of the adsorption of a flexible peptide onto a charged substrate in the 'wrong side' of the isoelectric point (WSIP), i.e. when surface and peptide charges have the same sign. As a model system, we focus on the casein macropeptide (CMP), both in the aglycosylated (aCMP) and fully glycosydated (gCMP) forms. We model the substrate as a uniformly charged plane while CMP is treated as a bead-and-spring model including electrostatic interactions, excluded volume effects and acid/base equilibria. Adsorption coverage, aminoacid charges and concentration profiles are computed by means of Monte Carlo simulations at fixed pH and salt concentration. We conclude that for different reasons the CMP can be adsorbed to both positively and negatively charged surfaces in the WSIP. For negatively charged surfaces, WSIP adsorption is due to the patchy distribution of charges: the peptide is attached to the surface by the positively charged end of the chain, while the repulsion of the surface for the negatively charged tail is screened by the small ions of the added salt. This effect increases with salt concentration. Conversely, a positively charged substrate induces strong charge regulation of the peptide: the acidic groups are deprotonated, and the peptide becomes negatively charged. This effect is stronger at low salt concentrations and it is more intense for gCMP than for aCMP, due to the presence of the additional sialic groups in gCMP.

• MeSH
• adsorpce MeSH
• cytidinmonofosfát MeSH
• izoelektrický bod MeSH
• kaseiny * MeSH
• peptidy * MeSH
• povrchové vlastnosti MeSH
• Publikační typ
• časopisecké články MeSH

Trifluralin is herbicide of the dinitroanilines group in which NO2 molecules are attached to the benzene ring at diverse positions. Trifluralin affects endocrine function and is listed as an endocrine disrupter in the European Union list. Therefore, its determination is so important in health science. In this study, an easy, sensitive and environmentally friendly method has been developed for determination of trifluralin based on its electrochemical oxidation on a three-electrode system designed on the surface of agricultural products using Ag-citrate/GQDs (graphene quantum dots) nano-ink. The sensor was prepared by direct writing on the surface of the samples. The designed electrodes were dried after 24 h at room temperature and used for trifluralin detection. Under optimized experimental conditions, the Ag-citrate/GQDs nano-ink based sensor was exhibited good sensitivity and specificity for trifluralin detection. The obtained linear range using the cyclic voltammetric (CV) technique is between 0.008 to 1 mM and low limit of quantification (LLOQ) was 0.008 mM. Also, the obtained linear range using differential pulse voltammetric (DPV) and square wave voltammetric (SWV) techniques is 0.005-0.04 mM with LLOQ of 0.005 mM. For further validation of the applicability of the proposed method, it was also used for detection of trifluralin on the surface of apple skin.

• Publikační typ
• časopisecké články MeSH

[This retracts the article DOI: 10.1016/j.heliyon.2020.e05779.].

• Publikační typ
• odvolání publikace MeSH

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.

• MeSH
• aktivace enzymů MeSH
• fenylalanin metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• karbohydrátdehydrogenasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• kinetika MeSH
• methionin metabolismus   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• oxidace-redukce MeSH
• peptidy chemie   MeSH
• peroxid vodíku metabolismus   MeSH
• sekvence aminokyselin MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• Trametes enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

New types of double-headed 2'-deoxycytidine 5'-O-triphosphates (dC(XC)TPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC(XC)TPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins.

• MeSH
• cytosin chemie   metabolismus   farmakologie   MeSH
• deoxycytosinnukleotidy chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• DNA-(cytosin-5-)methyltransferasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA biosyntéza   chemie   metabolismus   MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.

• MeSH
• ethylendichloridy metabolismus   MeSH
• halogenace MeSH
• hydrolasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• kumariny chemie   metabolismus   MeSH
• metylace MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• substrátová specifita MeSH
• Xanthobacter chemie   enzymologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH