Structural insight into the calcium ion modulated interdomain electron transfer in cellobiose dehydrogenase

AbstractCellobiose dehydrogenase (CDH) from wood degrading fungi represents a subclass of oxidoreductases with unique properties. Consisting of two domains exhibiting interdomain electron transfer, this is the only known flavocytochrome involved in wood degradation. High resolution structures of the separated domains were solved, but the overall architecture of the intact protein and the exact interface of the two domains is unknown. Recently, it was shown that divalent cations modulate the activity of CDH and its pH optimum and a possible mechanism involving bridging of negative charges by calcium ions was proposed. Here we provide a structural explanation of this phenomenon confirming the interaction between negatively charged surface patches and calcium ions at the domain interface

Ink-and-Ray: Bas-Relief Meshes for Adding Global Illumination Effects to Hand-Drawn Characters

We present a new approach for generating global illumination renderings of hand-drawn characters using only a small set of simple annotations. Our system exploits the concept of bas-relief sculptures, making it possible to generate 3D proxies suitable for rendering without requiring side-views or extensive user input. We formulate an optimization process that automatically constructs approximate geometry sufficient to evoke the impression of a consistent 3D shape. The resulting renders provide the richer stylization capabilities of 3D global illumination while still retaining the 2D handdrawn look-and-feel. We demonstrate our approach on a varied set of handdrawn images and animations, showing that even in comparison to ground truth renderings of full 3D objects, our bas-relief approximation is able to produce convincing global illumination effects, including self-shadowing, glossy reflections, and diffuse color bleeding

How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?

Inhibitors of the shikimate pathway are widely used as herbicides, antibiotics, and anti-infectious drugs. However, the regulation of the shikimic pathway is complex, and little is known about the feedback regulation of the shikimate dehydrogenase (SDH, EC 1.1.1.25) in plants. Thus, the aim of this study was to elucidate the kinetic mechanism of SDH purified from the root of Petroselinum crispum (parsley), to determine all possible reaction products and to identify phenylpropanoid compounds that affect its activity. Our results showed that the bisubstrate reaction catalyzed by P. crispum SDH follows a sequential ordered mechanism, except for three dead-end complexes. The main and lateral reactions of SDH were monitored by mass spectrometry, thereby detecting protocatechuic acid as a byproduct. Gallic acid was formed non-enzymatically, whereas quinate was not detected. Several polyphenolic compounds inhibited SDH activity, especially tannic, caffeic and chlorogenic acids, with IC50 0.014 mM, 0.15 mM, and 0.19 mM, respectively. The number of hydroxyl groups influenced their inhibition effect on SDH, and p-coumaric, t-ferulic, sinapic, syringic and salicylic acids were less effective SDH inhibitors. Nevertheless, one branch of the phenylpropanoid pathway may affect SDH activity through feedback regulation. © 2021 Elsevier LtdCharles University [SVV260572/2020]Univerzita Karlova v Praze, UK: SVV260572/202

Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10

<p>Abstract</p> <p>Background</p> <p>Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.</p> <p>Results</p> <p>A nitrilase gene was amplified by PCR from the cDNA library of <it>Aspergillus niger </it>K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in <it>Escherichia coli </it>BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val₃₂₇- Asn₃₅₆) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp₂₉₈-Val₃₁₃peptide was shortened to Asp₂₉₈-Arg₃₁₀in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.</p> <p>Conclusions</p> <p>The nitrilase from <it>Aspergillus niger </it>K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in <it>Aspergillus </it>and <it>Penicillium </it>genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by <it>in vitro </it>protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.</p

Spectroelectrochemistry at free-standing carbon nanotubes electrodes

A versatile and low-cost methodology for fabricating free-standing carbon nanotubes (CNT) electrodes for electrochemical and spectroelectrochemical applications is described. The uniformity, flexibility and resistance to bending of these films make them one of the most interesting membranes in a wide range of applications. CNT electrodes were characterized by Raman spectroscopy and scanning electron microscopy and their electrochemical performance was assessed employing various redox species such as ferrocenemethanol, hexacyanoferrate (II) and dopamine. Free-standing single-walled CNT electrodes exhibit good conductivity and transparency to UV–vis radiation, making them suitable as optically transparent electrodes. This is exemplified by monitoring, using UV–vis absorption spectroelectrochemistry, the electrodeposition of gold nanoparticles (AuNPs) on one face of the free-standing CNT electrodes, while the other face remained unmodifiedMinisterio de Economía y Competitividad (CTQ2014-55583-R, CTQ2014-61914-EXP, CTQ2015-71955-REDT) and Junta de Castilla y León (BU033U16)Ministerio de Economía y Competitividad (CTQ2014-55583-R, CTQ2014-61914-EXP, CTQ2015-71955-REDT) and Junta de Castilla y León (BU033U16

14. mezinárodní vědecká konference Didaktická konference 2021

Title in English: 14th International Scientific Conference Didactic Conference 2021: Proceedings Didactics of mathematics, natural sciences and vocational education represent developing areas and are a breeding ground for new ideas and approaches. In mathematics and physics, the proceedings provide an overview of the historical development of education process and the possibilities of activating students. From a practical point of view, physics and chemistry are interconnected by using simulations in teaching. Vocational education is represented from various perspectives, whether in technical education, by solving logistical problems, or educational activities in the teaching of economic subjects. Due to the current situation, there are abundant contributions on the effects of the ongoing pandemic on the educational process and distance learning

XXV. mezinárodní kolokvium o regionálních vědách

Title in English: 25th International Colloquium on Regional Sciences: Conference proceedings The conference proceedings consists of papers presented at the 25th International Colloquium on Regional Sciences that was organized by Department of Regional Economics and Administration FEA MU. It contains 57 articles arranged by topic. The individual articles deal with e.g. socioeconomic disparities among regions, regional policy, territory attractiveness, tourism, or regional public administration