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Chembiochem: a European Journal of Chemical Biology

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https://www.readbyqxmd.com/read/28514494/covalent-protein-labeling-by-spytag-spycatcher-in-fixed-cells-for-super-resolution-microscopy
#1
Veronica Pessino, Yemima Rose Citron, Siyu Feng, Bo Huang
Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. While the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy...
May 17, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28510317/efflux-pumps-may-not-be-the-major-drivers-of-qac-resistance-in-methicillin-resistant-staphylococcus-aureus
#2
Megan C Jennings, Megan E Forman, Stephanie M Duggan, Kevin P C Minbiole, William M Wuest
Quaternary ammonium compounds (QACs) are commonly used antiseptics that are now known to be subject to bacterial resistance. The prevalence and mechanisms of such resistance, however, remain underexplored. We investigated a variety of QACs, including those with multicationic structures (multiQACs), and the resistance displayed by a variety of Staphylococcus aureus strains with and without genes encoding efflux pumps, the purported main driver of bacterial resistance in MRSA. Through MIC, kinetic, and efflux-based assays, we find that neither the qacR/qacA system present in S...
May 16, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28509371/quantitative-assessment-of-sialo-glycoproteins-and-n-glycans-during-cardiomyogenic-differentiation-of-human-induced-pluripotent-stem-cells
#3
Sarah Anna Konze, Samanta Cajic, Astrid Oberbeck, René Hennig, Andreas Pich, Erdmann Rapp, Falk Buettner
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) may be used in regenerative medicine for individualized tissue transplants in future. For application in patients, the generated CMs have to be highly pure and well characterized. To overcome the prevalent scarcity of CM-specific markers, we quantitatively assessed cell surface exposed sialo-glycoproteins and N-glycans of hiPSCs, CM progenitors and CMs derived thereof. Applying a combination of metabolic labeling and specific sialo-glycoprotein capture, we could highly enrich and quantify membrane proteins during cardiomyogenic differentiation...
May 16, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28504873/controlling-the-regioselectivity-of-baeyer-villiger-monooxygenases-by-mutation-of-active-site-residues
#4
Kathleen Balke, Marcus Bäumgen, Uwe Bornscheuer
Baeyer-Villiger monooxygenase (BVMO) mediated regiodivergent conversions of asymmetric ketones can lead to the formation of "normal" or "abnormal" lactones. In a previous study we were able to change the regioselectivity of a BVMO by mutation of active site residues to smaller amino acids and thus creating more space. In this study we demonstrate, that this method can be used for other BVMO/substrate combinations as well. We investigated the regioselectivity of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase from Pseudomonas putida (OTEMO) for cis-bicyclo[3...
May 15, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28503897/aggregate-formation-of-oligonucleotides-that-assisted-molecular-imaging-for-tracking-of-oxygen-status-in-tumor-tissue
#5
Kazuki Yoshihara, Kohei Takagi, Aoi Son, Ryohsuke Kurihara, Kazuhito Tanabe
The use of DNA aggregates could be a promising strategy for molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. We prepared DNA-ruthenium conjugate (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the present DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner...
May 14, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28503789/exploring-the-structural-space-of-galectin-1-ligand-interaction
#6
Nadja Bertleff-Zieschang, Julian Bechold, Clemens Grimm, Michael Reutlinger, Petra Schneider, Gisbert Schneider, Juergen Seibel
Galectin-1 is a tumor-associated protein which, recognizing the Galβ1-4GlcNAc motif of cell surface glycoconjugates. Here, we report on the stepwise expansion of a multifunctional natural scaffold based on N-acetyllactosamine (LacNAc). We obtained a LacNAc mimetic equipped with an alkyne function on the 3'-hydroxyl group of the disaccharide facing towards a binding pocket adjacent to the carbohydrate recognition domain. It served as an anchor motif for further expansion via Sharpless-Huisgen-Meldal reaction, resulting in ligands with an almost identical binding mode to the natural carbohydrate template...
May 14, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28493500/conformational-analysis-of-the-mannosidase-inhibitor-kifunensine-a-quantum-mechanical-and-structural-approach
#7
Alexandra Males, Lluis Raich, Spencer J Williams, Carme Rovira, Gideon John Davies
The varied yet family-specific conformational pathways utilized by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tight binding and specific enzyme inhibitors. A cardinal example of a GH family specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low nanomolar inhibitor selective for GH family 47 inverting a-mannosidases. Here we show, through quantum mechanical approaches, that kifunensine is restrained to a 'ring-flipped' 1C4 conformation with another accessible, but higher-energy, region around the 1,4B conformation...
May 11, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28489326/highly-selective-stable-isotope-labeling-of-histidine-residues-using-a-novel-precursor-in-e-coli-based-overexpression-systems
#8
Julia Schörghuber, Leonhard Geist, Gerald Platzer, Robert Konrat, Roman Lichtenecker
The importance of NMR spectroscopy in unraveling the structural and dynamic properties of proteins is ever-expanding due to progress in experimental techniques, hardware development and novel labeling approaches. Multiple sophisticated methods of aliphatic residue labeling can be found in the literature, whereas the selective incorporation of NMR active isotopes into other amino acids still holds the potential for improvement. In order to close this methodical gap, we present a novel metabolic precursor for cell-based protein overexpression to assemble 13C/2H isotope patterns in backbone, as well as in side chain positions of the mechanistically distinguished histidine residue...
May 10, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28489325/reducing-macro-and-microheterogeneity-of-n-glycans-enabled-solving-the-crystal-structure-of-the-lectin-and-egf-like-domains-of-human-l-selectin-at-1-9-%C3%A3-resolution
#9
Stefanie Wedepohl, Jens Dernedde, Ardeschir Vahedi-Faridi, Rudolf Tauber, Wolfram Saenger, Haydar Bulut
L-selectin is a cell adhesion receptor located on the surface of most leukocytes and contains a total of 7 N-glycosylation sites. To obtain the crystal structure of human L-selectin, we expressed a shortened version comprising the C-type lectin and EGF-like domains of L-selectin (termed LE hereafter) and systematically analysed mutations of the three glycosylation sites at Asn22, Asn66, and Asn139 in order to reduce macroheterogeneity. After we further removed microheterogeneity, we obtained crystals which diffracted X-rays up to 1...
May 10, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28489306/pipecolic-acid-hydroxylases-a-monophyletic-clade-among-cis-selective-bacterial-proline-hydroxylases-that-discriminates-l-proline
#10
Johanna Mattay, Wolfgang Hüttel
Proline hydroxylases are Fe(II)/2-oxoglutarate-dependent enzymes that hydroxylate L-proline and derivatives, such as L-pipecolic acid, the six-membered ring homologue of L proline. We have established that there is a distinct group of conserved bacterial enzymes that hydroxylate L-pipecolic acid and trans-3- and trans-4-methyl-L-proline, but virtually no L proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent L-proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp...
May 10, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28488816/characterization-of-a-methyltransferase-almcii-in-chalcomycin-biosynthesis-the-first-o-methyltransferase-in-tylf-family-works-on-a-4-deoxysugar
#11
Ping Dai, Chuan-Xi Wang, Hao Gao, Qiao-Zhen Wang, Xiao-Long Tang, Guo-Dong Chen, Kui Hong, Dan Hu, Xin-Sheng Yao
Sugar O-methylation is a ubiquitous modification in natural products and has diverse roles. This realization has inspired many attempts to search for novel methyltransferases. Chalcomycins are a group of 16-membered macrolides containing two methylated sugars, which requires three methyltransferases for their biosynthesis. Here, we identified that AlmCII, a sugar O-methyltransferase belonging to the TylF family which thus far only methylates sugars with a 4'-hydroxyl group, can methylate a 4',6'-dideoxysugar in the biosynthesis of chalcomycins...
May 10, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28485853/1-2-4-triazine-modified-2-deoxyuridine-triphosphate-for-efficient-bioorthogonal-fluorescent-labeling-of-dna
#12
Krisana Peewasan, Hans-Achim Wagenknecht
In order to establish the Diels-Alder reaction with inverse electron demand for postsynthetic DNA modification a 1,2,4-triazine-modified 2'-deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4-triazine group was attached at the 5-position of 2'-deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions using a mixture of dATP, dGTP, dCTP and the modified 2'-deoxyuridine triphosphate with the KOD XL and Vent polymerases...
May 9, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28474822/in-vitro-biochemical-assays-for-o-glcnac-processing-enzymes
#13
Eun Ju Kim
O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are the only enzymes that regulate the dynamics of protein O-GlcNAcylation. Protein O-GlcNAcylation is an important post-translational modification (PTM) of nuclear and cytoplasmic proteins with O-linked ß-N-acetyl-glucosamine (O-GlcNAc). O-GlcNAc and its enzymes are involved in a wide variety of cellular processes and are linked to the pathological progression of chronic diseases. Considering their emerging biological significance, systematic and rapid methods to determine the activities of OGT and OGA have become essential and several chemical/biochemical methods for measuring these enzymes' activities have been developed...
May 5, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28474804/active-site-his85-of-pasteurella-dagmatis-sialyltransferase-facilitates-productive-sialyl-transfer-and-so-prevents-futile-hydrolysis-of-cmp-neu5ac
#14
Katharina Schmölzer, Manuel Eibinger, Bernd Nidetzky
Sialyltransferases of glycosyltransferase family GT-80 are considered as multifunctional due to an array of activities detected. They exhibit glycosyl transfer, trans-sialylation and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3-sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200-fold less efficient than the wild type for sialylation of lactose and exhibited relaxed site selectivity to form not only α2,3 but also α2,6-sialylated product (21%)...
May 5, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28472541/exploring-and-exploiting-acceptor-preferences-of-the-human-polysialyltransferases-as-a-basis-for-an-inhibitor-screen
#15
Jörg Ehrit, Timothy G Keys, Mark Sutherland, Chris Meier, Saskia Wolf, Robert Falconer, Rita Gerardy-Schahn
α2,8 linked polysialic acid (polySia) is an oncofetal antigen with high abundance during embryonic development and reappearance in malignant tumors of neuroendocrine origin. Responsible for polySia biosynthesis are the two polysialyltransferases (polySTs) ST8SiaII & IV. During development, both enzymes are essential to control polySia expression. However, in the tumor situation ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for the development of novel cancer therapeutics...
May 4, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28471098/non-enzymatic-oligomerization-of-3-5-cyclic-cmp-induced-by-proton-and-uv-irradiation-hints-at-a-non-fastidious-origin-of-rna
#16
Giovanna Costanzo, Alessandra Giorgi, Anita Scipioni, Anna Maria Timperio, Carmine Mancone, Marco Tripodi, Michail Kapralov, Eugene Krasavin, Holger Kruse, Jiri Sponer, Judit E Sponer, Vaclav Ranc, Michal Otyepka, Samanta Pino, Ernesto Di Mauro
We report that 3',5' cyclic CMP undergoes non-enzymatic di- and trimerization at 20°C under dry conditions upon proton- or UV irradiation. The reaction involves stacking of the cyclic monomers and subsequent polymerization by serial transphosphorylations between the stacked monomers. Proton- and UV-induced oligomerization of 3',5' cyclic CMP demonstrates that, in addition to purines, also pyrimidines may have taken part in the spontaneous generation of RNA in plausible prebiotic conditions as well as in an extraterrestrial context...
May 4, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28470863/fluoro-no-a-nitric-oxide-donor-with-a-fluorescence-reporter
#17
Govindan Ravikumar, Meisam Bagheri, Deepak K Saini, Harinath Chakrapani
Nitric oxide (NO) plays significant signalling roles in cells and controlled generation of NO is of therapeutic relevance. Although a number of methodologies for delivery and detection of nitric oxide (NO) are available, these events are typically mutually exclusive. Furthermore, the efficiency of delivery of NO can be compromised by detection technologies that consume NO during the detection event. Here, we report FLUORO/NO, an esterase activated diazeniumdiolate-based NO donor with an in-built fluorescence reporter...
May 4, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28470883/selective-covalent-protein-modification-by-4-halopyridines-through-catalysis
#18
Christopher Schardon, Alfred Tuley, Joyce Er, Jake Swartzel, Walter Fast
4-Halopyridines are developed herein as selective, tunable, and "switchable" covalent protein modifiers for use in the development of chemical probes. Non-enzymatic reactivity of 4-chloropyridine with amino acids and thiols is ranked with respect to common covalent protein modifying reagents and found to have reactivity similar to that of acrylamide, but can be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse fragment-sized 4-halopyridines inactivate human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active-site Cys, acting as quiescent affinity labels that require "off pathway" catalysis via a stabilization of the protonated pyridinium by a neighboring Asp residue...
May 3, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28470825/three-in-one-go-thermo-solvent-and-catalytic-stability-by-engineering-the-cofactor-binding-element-of-amine-transaminases
#19
Tim Börner, Sebastian Rämisch, Sebastian Bartsch, Andreas Vogel, Patrick Adlercreutz, Carl Grey
Amine transaminases (ATA) catalyse enantioselectively the direct amination of ketones, but the insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from a substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring binding element that also shapes the active site entrance. Only two point mutations in this motif improved thermo- and operational stability in both biphasic media and neat organic solvent...
May 3, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28464395/specificity-effects-of-amino-acid-substitutions-in-promiscuous-hydrolases-context-dependence-of-catalytic-residue-contributions-to-local-fitness-landscapes-in-nearby-sequence-space
#20
Christopher D Bayer, Bert van Loo, Florian Hollfelder
Catalytic promiscuity can facilitate evolution of enzyme functions-a multifunctional catalyst may act as a springboard for efficient functional adaptation. We test the effect of single mutations on multiple activities in two groups of promiscuous AP superfamily members to probe this hypothesis. We quantify the effect of site-saturating mutagenesis of an analogous, nucleophile-flanking residue in two superfamily members: an arylsulfatase (AS) and a phosphonate monoester hydrolase (PMH). Statistical analysis suggests that no one physicochemical characteristic alone explains the mutational effects...
May 2, 2017: Chembiochem: a European Journal of Chemical Biology
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