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    High-throughput-feasible screening tool for determining enzyme stabilities against organic solvents directly from crude extracts.
    Chembiochem 2017 Oct 12. Epub 2017 Oct 12.
    Universität Bielefeld, Fakultät für Chemie Organische Chemie I, Universitätsstr. 25, 33615, Bielefeld, GERMANY.
    Biotransformations in organic chemistry frequently suffer from limitations caused by low water-solubility of substrates and product inhibition. Both, usually are addressed by the addition of organic co-solvents, which often accompanies at the expense of enzyme stability. A common method for measuring enzyme stabilities is the determination of the enzyme´s melting temperature (Tm). Read More

    Orthogonal expression of an artificial metalloenzyme for abiotic catalysis.
    Chembiochem 2017 Oct 11. Epub 2017 Oct 11.
    UNC Chapel Hill Undergraduate student, UNITED STATES.
    A cytochrome P450 was engineered to selectively incorporate Ir(Me)-deuteroporphyrin IX (Ir(Me)-DPIX), in lieu of heme, in bacterial cells. Cofactor selectivity was altered by introducing mutations within the heme-binding pocket that discriminate the deuteroporphyrin macrocycle, in combination with mutations to the P450 axial cysteine to accommodate a pendant methyl group on the Ir(Me)-center. This artificial metalloenzyme was investigated for activity in non-native metallocarbenoid-mediated olefin cyclopropanation reactions and showed enhanced activity for aliphatic and electron-deficient olefins when compared to the native heme enzyme. Read More

    Regulating DNA Self-Assembly by DNA-Surface Interactions.
    Chembiochem 2017 Oct 11. Epub 2017 Oct 11.
    Purdue University, Chemistry, 560 Oval Drive, 47907, West Lafayette, UNITED STATES.
    DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and only involves DNA-DNA interactions. When confined onto surfaces, DNA-surface interaction becomes an additional, important factor to DNA self-assembly. Read More

    Development of a sensitive microarray platform for the ranking of galectin inhibitors: identification of a selective galectin-3 inhibitor.
    Chembiochem 2017 Oct 11. Epub 2017 Oct 11.
    Universite de Nantes, UFIP UMR CNRS 6286, 2 rue de la Houssinière, BP92208, 44322, Nantes Cedex 3, FRANCE.
    Glycan microarrays are useful tools for lectin glycan profiling. The use of glycan microarray based on evanescent-field fluorescence detection is herein further extended to the screening of lectin inhibitors in competitive experiments. Efficacy of this approach has been tested with 2/3' mono- or di-aromatic type II lactosamine derivatives and galectins as targets and validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. Read More

    PREREQUISITES OF ISOPEPTIDE BOND FORMATION IN MICROCYSTIN BIOSYNTHESIS.
    Chembiochem 2017 Oct 11. Epub 2017 Oct 11.
    University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Str. 24/25, 14476, Potsdam-Golm, GERMANY.
    Biosynthesis of the potent cyanobacterial hepatotoxin microcystin involves isopeptide bond formation via the carboxylic acid side chains of D-glutamate and β-methyl- D-aspartate. Analysis of the in vitro activation profiles of the two corresponding adenylation domains McyE-A and McyB-A2 either in a didomain or a tridomain context with the cognate thiolation and the upstream condensation domain revealed that substrate activation of both domains strictly depends on the presence of the condensation domains. We further identified two key amino acids in the binding pockets of both adenylation domains that could serve as a bioinformatic signature of isopeptide bond forming modules incorporating D-glutamate or D-aspartate. Read More

    Enzymatic incorporation of modified purine nucleotides in DNA.
    Chembiochem 2017 Oct 11. Epub 2017 Oct 11.
    Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49 - box 1030, 3000, Leuven, BELGIUM.
    A series of nucleotide analogues, with a hypoxanthine base moiety (8-amino-hypoxanthine, 1-methyl-8-amino-hypoxanthine and 8-oxo-hypoxanthine) together with 5-methyl-isocytosine were tested as potential pairing partners of N8-glycosylated nucleotides with an 8-azaguanine and 8-aza-9-deazaguanine base moiety, using DNA polymerases (incorporation studies). The best results were obtained with the 5-methyl-isocytosine nucleotide followed by the 1-metyl-8-amino-hypoxanthine nucleotide. The experiments demonstrate that a) small differences in the structure (8-azaguanine versus 8-aza-9-deazaguanine) might lead to significant differences in recognition efficiency and selectivity b) base pairing via Hoogsteen recognition, at the polymerase level is possible c) 8-aza-9-deazaguanine represents a self-complementary base pair and d) a correlation exists between the in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions are observed). Read More

    A Conjugate of an anti-EGFR VHH and a Cell-penetrating Peptide Drives Receptor Internalization and Blocks EGFR Activation.
    Chembiochem 2017 Oct 10. Epub 2017 Oct 10.
    Radboudumc, Pathology, Geert Grooteplein-Zuid 10, 6525 GA, Nijmegen, NETHERLANDS.
    Overexpression of (mutated) receptor tyrosine kinases is a characteristic of many aggressive tumors and induction of receptor uptake has long been recognized as a therapeutic modality. A conjugate of a synthetically produced cell penetrating peptide, corresponding to amino acids 38-59 of human lactoferrin, and the recombinant llama single-domain antibody (VHH) 7D12 that binds the human epidermal growth factor receptor (EGFR), was generated via sortase A-mediated transpeptidation. The conjugate blocks EGF-mediated EGFR activation with higher efficacy than both modalities alone, a phenomenon that is caused by both effective receptor blockade and internalization. Read More

    Sustainable and continuous synthesis of enantiopure L-amino acids using a versatile immobilised multi-enzyme system.
    Chembiochem 2017 Oct 9. Epub 2017 Oct 9.
    CIC biomaGUNE, Paseo Miramon 182, 20009, San Sebastian, SPAIN.
    The enzymatic synthesis of -amino acids appears as a sustainable and efficient alternative to chemical processes, where achieving enantiopure products is a hard task. To more efficiently adress this synthesis, we desinged and fabricate a hierarchical architecture that irreversibly co-immobilises an amino acid dehydrogenase with polyethyleneimine on porous agarose beads. The cationic polymer acts as an irreversible anchoring layer for the formate dehydrogenase. Read More

    Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates.
    Chembiochem 2017 Oct 7. Epub 2017 Oct 7.
    Universität Göttingen, Organische und Biomolekulare Chemie, Tammannstr. 2, 37077, Göttingen, GERMANY.
    A novel peptide-peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating in general allows for various kind of covalent ligation reactions. Read More

    Electron Bifurcation Makes the Puzzle Pieces Fall Energetically into Place in Methanogenic Energy Conservation.
    Chembiochem 2017 Oct 6. Epub 2017 Oct 6.
    Washington State University, Institute of Biological Chemistry, 287 Clark Hall, 99164, Pullman, UNITED STATES.
    Microbial life has evolved a wide range of metabolisms exploiting in many cases unanticipated suites of oxidation-reduction reactions to generate energy. Although many of these suites of reactions don't allow these microbes to enjoy the same quality of energetic life that we enjoy via respiration/oxidative phosphorylation, it has conferred the ability for life to exploit almost any oxidation-reduction reaction. In many of these cases when energy is sparing, the difference between life and death may be conserving the maximal amount of energy and minimizing loss of free energy through heat. Read More

    Labelling of Proteins with Carbon Nanodots.
    Chembiochem 2017 Oct 6. Epub 2017 Oct 6.
    Indian Institute of Technology Mandi, School of Basic Sciences, Academic Block, 175001, Mandi, INDIA.
    We present efficient labelling of several proteins with orange emissive carbon dots. N-Hydroxysuccinimide was used to activate the carboxyl groups of carbon dots which subsequently reacted with lysine groups present on the protein. The labelling was confirmed by UV-absorption spectroscopy, poly-acrylamide gel electrophoresis, and fluorescence correlation spectroscopy. Read More

    Towards tuneable retaining glycosidase-inhibiting peptides by mimicry of a plant flavonol warhead.
    Chembiochem 2017 Oct 6. Epub 2017 Oct 6.
    Utrecht Institute for Pharmaceutical Sciences, Chemical Biology and Drug Discovery, Universiteitsweg 99, 3584CG, Utrecht, NETHERLANDS.
    Retaining glycosidases are an important class of enzymes involved in glycan degradation. In order to better study the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors each specific to a particular glycosidase would be an invaluable toolkit. Towards this goal, we detail here a more in-depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α-amylase (HPA). Read More

    Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds.
    Chembiochem 2017 Oct 5. Epub 2017 Oct 5.
    Technische Universität Wien, Institut für Angewandte Synthesechemie, Getreidemarkt 9, 1060, Wien, AUSTRIA.
    We investigated a synthetic cascade for the transformation of primary alcohols into polyhydroxylated compounds in Escherichia coli by the in situ preparation of cytotoxic aldehyde intermediates and subsequent aldolase mediated C-C bond formation. An enzymatic toolbox consisting of the alcohol dehydrogenase (ADH) AlkJ from Pseudomonas putida and the dihydroxyacetone/hydroxyacetone (DHA/HA) accepting aldolase variant Fsa1-A129S was applied. Pathway optimization was performed on genetic and process level. Read More

    AGT Activity Towards Intrastrand Cross-linked DNA is Modulated by the Alkylene Linker.
    Chembiochem 2017 Oct 5. Epub 2017 Oct 5.
    Concordia University, Department of Chemistry and Biochemistry, 7141 Sherbrooke St. West, H4B 1R6, Montreal, CANADA.
    DNA oligomers containing dimethylene and trimethylene intrastrand cross-links (IaCL) between the O4- and O6-atoms of neighboring thymidine (T) and 2´-deoxyguanosine (dG) residues have been prepared by solid-phase synthesis. UV thermal denaturing (Tm) experiments revealed that these IaCL had a destabilizing effect on the DNA duplex relative to the control. Circular dichroism spectroscopy suggested these IaCL induced minimal structural distortions. Read More

    Simultaneous IR-spectroscopic observation of α-synuclein, lipids, and solvent reveals an alternative membrane-induced oligomerization pathway.
    Chembiochem 2017 Oct 5. Epub 2017 Oct 5.
    University of Konstanz, Chemistry, Universitätsstr. 10, 78457, Konstanz, GERMANY.
    The intrinsically disordered protein α-synuclein (αS), a known pathogenic factor for Parkinson's disease, can adopt defined secondary structures when interacting with membranes or during fibrillation. The αS-lipid interaction and the implications of this process for aggregation and damage to membranes are still poorly understood. Therefore, we established a label-free infrared (IR) spectroscopic approach to simultaneously monitor αS conformation and membrane integrity. Read More

    Protonation-initiated cyclization by a class II terpene cyclase assisted by tunneling.
    Chembiochem 2017 Oct 5. Epub 2017 Oct 5.
    KTH Royal Institute of Technology, School of Chemical Science and Engineering, Science for Life Laboratory, Tomtebodavägen 23B, 171 65, Solna, 17165, Stockholm, SWEDEN.
    Terpenes represent one of the most diversified classes of natural products with potent biological activities. The key to the myriad of polycyclic terpene skeletons with crucial functions in organisms from all kingdoms of life are terpene cyclase enzymes. These biocatalysts enable stereospecific cyclization of relatively simple, linear pre-folded polyisoprenes by highly complex, partially concerted, electrophilic cyclization cascades that remain incompletely understood. Read More

    Genomic studies reveal new aspects of the biology of DNA damaging agents.
    Chembiochem 2017 Oct 3. Epub 2017 Oct 3.
    University of Basel, Department of Chemistry, St. Johanns-Ring 19, 4056, Basel, SWITZERLAND.
    A flurry of papers have appeared recently that forces a rethinking of our understanding of how chemicals, light, and metal-complexes damage our genomes. The conventional wisdom was that damaging agents are indiscriminate and it was statistical bad luck coupled with evolutionary selection that drove mutational signatures after exposure of DNA to damaging agents. Recent data, however, suggests that primary DNA damage itself does not drive mutational signatures, instead it is the selectivity of repair pathways on different regions of the genome that is decisive. Read More

    Determination of alkyl-donor promiscuity of tyrosine-O-prenyltransferase SirD from Leptosphaeria maculans.
    Chembiochem 2017 Sep 27. Epub 2017 Sep 27.
    University of Oklahoma, Chemistry and Biochemistry, 101 Stephenson Pkwy, SLSRC room 2190, 73019, United States, 73019, Norman, UNITED STATES.
    Natural product prenyltransferases are known to display relaxed acceptor substrate specificity. While recent studies using a small set of unnatural alkyl donors reveal prenyltransferases to be flexible towards their alkyl donors, the scope of alkyl donor specificity remains poorly understood. Towards this goal, we report the synthesis of 20 unnatural alkyl pyrophosphate donors and assessment of the tyrosine O-prenyltransferase SirD catalyzed reaction with synthetic unnatural alkyl pyrophosphate analogues. Read More

    A high-throughput mass spectrometry-based assay for identifying biochemical function of putative glycosidases.
    Chembiochem 2017 Sep 27. Epub 2017 Sep 27.
    Indiana University, Department of Chemistry, 120A Simon Hall, 212 S. Hawthorne Drive, 47405, Bloomington, UNITED STATES.
    The most common glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position of potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Read More

    The Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding.
    Chembiochem 2017 Sep 26. Epub 2017 Sep 26.
    Massachusetts Institute of Technology, Chemistry, Francis Bitter Magnet Laboratory, 170 Albany Street, NW14-5107, 02139, Cambridge, UNITED STATES.
    The impact of the incorporation of a non-natural amino acid (NNAA) onto protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post-translationally in vivo and in vitro using click-chemistry. Here, we present a structural characterization of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 using NMR spectroscopy and X-ray crystallography. Read More

    Harzianone Biosynthesis by the Biocontrol Fungus Trichoderma.
    Chembiochem 2017 Sep 25. Epub 2017 Sep 25.
    Universität Bonn, Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Straße 1, 53121, Bonn, GERMANY.
    Analysis of the volatile terpenes produced by seven fungal strains of the genus Trichoderma by use of a closed-loop stripping apparatus (CLSA) revealed a common production of harzianone, a bioactive, structurally unique diterpenoid consisting of a fused tetracyclic 4,5,6,7-membered ring system. The terpene cyclization mechanism was studied by feeding experiments using selectively 13C- and 2H-labeled synthetic mevalonolactone isotopologues, followed by analysis of the incorporation patterns by 13C-NMR spectroscopy and GC/MS. The structure of harzianone was further supported from a 13C,13C-COSY experiment of the in vivo generated fully 13C-labeled diterpene. Read More

    Gb3 Glycosphingolipids with Fluorescent Oligoene Fatty Acids: Synthesis and Phase Behavior in Model Membranes.
    Chembiochem 2017 Sep 20. Epub 2017 Sep 20.
    TU Braunschweig, Institut für Organische Chemie, Hagenring 30, 38106, Braunschweig, Germany.
    Glycosphingolipids are involved in a number of physiological and pathophysiological processes, and they serve as receptors for a variety of bacterial toxins and viruses. To investigate their function in lipid membranes, fluorescently labeled glycosphingolipids are highly desirable. Herein, a synthetic route to access Gb3 glycosphingolipids with fluorescently labeled fatty acids, consisting of pentaene and hexaene moieties either at the terminus or in the middle of the acyl chain, has been developed. Read More

    Dodging Endosomes: Effective Cytosolic Antibody Delivery.
    Chembiochem 2017 Sep 21. Epub 2017 Sep 21.
    Radboud University Nijmegen, Institute for Molecules and Materials, Bio-organic Chemistry, Heyendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands.
    On the inside: New methodologies for delivering antibodies right into the cytosol of cells either directly across the plasma membrane or by allowing the antibody to escape from endosomes have been proposed recently by the Cardoso/Hackenberger and Futaki groups, respectively. Read More

    Tandem biocatalysis unlocks the challenging de novo production of plant natural products.
    Chembiochem 2017 Sep 20. Epub 2017 Sep 20.
    CNRS, UMR8172 EcoFoG, AgroParisTech, Cirad, INRA, Université des Antilles, Université de Guyane, FRANCE.
    Bioengineering the genes of plants and microbes is becoming a very competitive strategy in synthesis to produce pharmacologically relevant plant natural products and their unnatural analogues. During the last 20 years several plant genes have been identified, cloned and expressed successfully unlocking de novo production of plant metabolites such as morphine, strictosidine, artemisinin, taxol® and resveratrol. Future development for accelerating the discovery of gene candidates relies on innovative computational methods combined with transcriptomics and metabolomics analysis to make plant genome mining more feasible. Read More

    Continuous Production of Ursodeoxycholic Acid using Two Cascade Reactors with Coimmobilized Enzymes.
    Chembiochem 2017 Sep 19. Epub 2017 Sep 19.
    East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering, 130 Meilong Road, Shanghai 200237, China, 200237, Shanghai, CHINA.
    Ursodeoxycholic acid (UDCA) is an effective drug for the treatment of hepatitis. In this study, 7α-hydroxysteroid dehydrogenase (7α-HSDH) and lactate dehydrogenase (LDH), as well as 7β-HSDH and glucose dehydrogenase (GDH), were co-immobilized onto an epoxy-functionalized resin (ES-103) for catalyzing the synthesis of UDCA from chenodeoxycholic acid (CDCA). Through optimizing the immobilization pH, time and the loading ratio of enzymes to resin, the specific activities of immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 were 43. Read More

    Biosynthetic machinery of diterpene pleuromutilin isolated from basidiomycete fungi.
    Chembiochem 2017 Sep 19. Epub 2017 Sep 19.
    Hokkaido University, Department of Chemistry, Graduate School of Science,, Kita-ku Kita 10 Jo Nishi 8 Chome, 060-0810, Sapporo, JAPAN.
    The diterpene pleuromutilin is a ribosome-targeting antibiotic isolated from basidiomycete fungi, such as Clitopilus pseudo-pinsitus. Here, we report the functional characterization of all biosynthetic enzymes, involved in pleuromutilin biosynthesis, and propose a biosynthetic pathway. In vitro enzymatic reactions and mutational analysis revealed that a labdane-related diterpene synthase, Ple3, catalyzes two rounds of cyclization from geranylgeranyl diphosphate to premutilin possessing a characteristic 5-6-8-tricyclic carbon skeleton. Read More

    A Systematic Structure Activity Study of a New Type of Small Peptidic Transfection Vectors Reveals the Importance of a Special Oxo-Anion Binding Motif for Gene Delivery.
    Chembiochem 2017 Sep 15. Epub 2017 Sep 15.
    Universität Duisburg-Essen, Institut für Organische Chemie Fachbereich Chemie, Universitätsstraße 5, 45141, Essen, GERMANY.
    We discovered a new class of artificial peptidic transfection vectors based on an artificial anion binding motif, the guanidiniocarbonylpyrrole cation (abbreviated as GCP). This new type of vector is surprisingly small compared to traditional systems and our previous work suggested that the GCP group is important for promoting the critical endosomal escape. We now present here a systematic comparison of similar DNA-ligands featuring our GCP oxo-anion binding motif with DNA-ligands only consisting of natural occurring amino acids. Read More

    Mechanistic insights into cyclic peptide generation by DnaE split-inteins through quantitative and structural investigation.
    Chembiochem 2017 Sep 15. Epub 2017 Sep 15.
    TU Munich Chair of Biochemistry, Chemistry, Lichtenbergstr. 4, 85747, Garching, GERMANY.
    Inteins carry out protein-splicing reactions, which are utilised in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in a head-to-tail cyclisation of the target sequence, which can be used to genetically encode and express libraries of cyclic peptides. The efficiency of the splicing reaction depends on the target sequence. Read More

    Bioluminescence Resonance Energy Transfer (BRET) Coupled Annexin V Functionalized Quantum Dots for Near-Infrared Optical Detection of Apoptotic Cells.
    Chembiochem 2017 Sep 13. Epub 2017 Sep 13.
    JAPAN.
    Deregulation in apoptosis induces numerous diseases such as cancer, cardiovascular and neurodegenerative diseases. The detection of apoptotic cells is crucial for understanding the mechanism of those diseases and for therapy development. Although optical imaging using visible-emitting fluorescent probes such as FITC-labeled annexin V is widely used for the detection of apoptotic cells, there are a very limited probes that can be used in near-infrared (NIR) region over 700 nm. Read More

    Synthesis and Multiple Incorporations of 2'-O-Methyl-5-hydroxymethylcytidine, 5-Hydroxymethylcytidine and 5-Formylcytidine Monomers into RNA Oligonucleotides.
    Chembiochem 2017 Sep 13. Epub 2017 Sep 13.
    Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
    The synthesis of 2'-O-methyl-5-hydroxymethylcytidine (hm(5) Cm), 5-hydroxymethylcytidine (hm(5) C) and 5-formylcytidine (f(5) C) phosphoramidite monomers has been developed. Optimisation of mild post-synthetic deprotection conditions enabled the synthesis of RNA containing all four naturally occurring cytosine modifications (hm(5) Cm, hm(5) C, f(5) C plus 5-methylcytosine). Given the considerable interest in RNA modifications and epitranscriptomics, the availability of synthetic monomers and RNAs containing these modifications will be valuable for elucidating their biological function(s). Read More

    Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants.
    Chembiochem 2017 Sep 12. Epub 2017 Sep 12.
    Institute of Biochemistry and Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
    Squalene-hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl-farnesyl ether product from farnesol. Read More

    Characterisation of the L-cystine-b-lyase PatB from Phaeobacter inhibens, an enzyme involved in the biosynthesis of the marine antibiotic tropodithietic acid.
    Chembiochem 2017 Sep 12. Epub 2017 Sep 12.
    Universität Bonn, Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Straße 1, 53121, Bonn, GERMANY.
    The L-cystine-b-lyase from Phaeobacter inhibens is involved in the biosynthesis of the sulfur containing antibiotic tropodithiethic acid. The recombinant enzyme was obtained by heterologous expression in Escherichia coli and biochemically characterised by unambiguous chemical identification of the products formed from the substrate L-cystine, investigation of the substrate spectrum, determination of the enzyme kinetics, sequence alignment with closely related homologs and site-directed mutagenesis to identify a highly conserved lysine residue that is critical for functionality. PatB from P. Read More

    Fluorescent Probe DCVJ Shows High Sensitivity for Characterization of Amyloid β-Peptide Early in the Lag Phase.
    Chembiochem 2017 Sep 11. Epub 2017 Sep 11.
    Department of Physics and Astronomy, Michigan State University, 567 Wilson Road, Room 4227, East Lansing, MI, 48824, USA.
    The aggregation of intrinsically disordered and misfolded proteins in the form of oligomers and fibrils plays a crucial role in a number of neurological and neurodegenerative diseases. Currently, most probes and biophysical techniques that detect and characterize fibrils at high resolution fail to show sensitivity and binding for oligomers. Here, we show that 9-(dicyano-vinyl)julolidine (DCVJ), a class of molecular rotor, binds amyloid beta (Aβ) early aggregates, and we report the kinetics as well as packing of the oligomer formation. Read More

    Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein-Ligase System.
    Chembiochem 2017 Sep 10. Epub 2017 Sep 10.
    Department of Pathology and Laboratory Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
    Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive, as each new recombinant antibody needs to be optimized for expression, stability, bio-distribution, and pharmacokinetics. We have developed a new way to construct recombinant antibody-like "devices" by using a bottom-up approach to build them from well-behaved discrete recombinant antibody domains or "parts". Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation. Read More

    Creating Oxidase-Peroxidase Fusion Enzymes as a Toolbox for Cascade Reactions.
    Chembiochem 2017 Sep 8. Epub 2017 Sep 8.
    Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands.
    A set of bifunctional oxidase-peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Read More

    A Single-Framework Synthetic Antibody Library Containing a Combination of Canonical and Variable Complementarity Determining Regions.
    Chembiochem 2017 Sep 7. Epub 2017 Sep 7.
    University of Saskatchewan, Department of Pathology, 107 Wiggins Rd, S7N 5E5, Saskatoon, CANADA.
    Synthetic antibody libraries have been used to generate antibodies with favorable biophysical and pharmacological properties. Here, we describe the design, construction, and validation of a phage-displayed Fab library built on a modified trastuzumab framework with four fixed and two diversified complementarity-determining regions (CDRs). CDRs L1, L2, H1, and H2 were fixed to preserve the most-frequent 'canonical' CDR conformation preferred by the modified trastuzumab Fab framework. Read More

    Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.
    Chembiochem 2017 Sep 7. Epub 2017 Sep 7.
    Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 80/82, 6020, Innsbruck, Austria.
    The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitamin B12 ((57) Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Read More

    Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step.
    Chembiochem 2017 Oct 7;18(19):1910-1913. Epub 2017 Sep 7.
    Department of Applied Biological Chemistry, Faculty of Agriculture, and, Graduate School of Science and Technology, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata, 950-2181, Japan.
    Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl-β-curcumene (C35 ) but a bicyclic scaffold from squalene (C30 ) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. Read More

    Aromatic Halogenation by Using Bifunctional Flavin Reductase-Halogenase Fusion Enzymes.
    Chembiochem 2017 Sep 6. Epub 2017 Sep 6.
    Department of Chemistry, University of Chicago, 5735 South Ellis Avenue, SCL 302, Chicago, IL, 60637, USA.
    The remarkable site selectivity and broad substrate scope of flavin-dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. Read More

    A Diversity-Oriented Library of Fluorophore-Modified Receptors Constructed from a Chemical Library of Synthetic Fluorophores.
    Chembiochem 2017 Sep 6. Epub 2017 Sep 6.
    Institute of Advanced Energy, Kyoto University, Uji, Kyoto, 611-0011, Japan.
    The practical application of biosensors can be determined by evaluating the sensing ability of fluorophore-modified derivatives of a receptor with appropriate recognition characteristics for target molecules. One of the key determinants for successfully obtaining a useful biosensor is wide variation in the fluorophores attached to a given receptor. Thus, using a larger fluorophore-modified receptor library provides a higher probability of obtaining a practically useful biosensor. Read More

    Effect of Stapling Architecture on Physiochemical Properties and Cell Permeability of Stapled α-Helical Peptides: A Comparative Study.
    Chembiochem 2017 Sep 6. Epub 2017 Sep 6.
    Laboratory of Cytophysiology, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, 518055, China.
    Stapled peptides have emerged as a new class of targeting molecules with high binding affinity and specificity for intracellular undruggable targets. Their ability to penetrate cell membranes is exceptionally intriguing but remains elusively and controversially discussed. To understand the effect of stapling architectures on their physiochemical properties and to aid in promoting their cell permeability, we report herein a comparative study on the physiochemical properties and cell permeability of stapled α-helical peptides with different types of crosslinks. Read More

    Euphorbia tirucalli β-Amyrin Synthase: Critical Roles of Steric Sizes at Val483 and Met729 and the CH-π Interaction between Val483 and Trp534 for Catalytic Action.
    Chembiochem 2017 Sep 5. Epub 2017 Sep 5.
    Graduate School of Science and Technology and, Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata, 950-2181, Japan), E-mail: address.
    The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli β-amyrin synthase were revealed by comparing the enzyme activities of site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 predominantly afforded monocyclic camelliol C, which suggested that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair-chair-chair-boat-boat conformation. The Ile variant, with a somewhat larger bulk, afforded β-amyrin as the dominant product. Read More

    Visualizing the Adenylation Activities and Protein-Protein Interactions of Aryl Acid Adenylating Enzymes.
    Chembiochem 2017 Sep 4. Epub 2017 Sep 4.
    Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan.
    Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Read More

    Identification of Chimeric αβγ Diterpene Synthases Possessing both Type II Terpene Cyclase and Prenyltransferase Activities.
    Chembiochem 2017 Sep 4. Epub 2017 Sep 4.
    Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
    Two unusual diterpene synthases composed of three domains (α, β, and γ) were identified from fungal Penicillium species. They are the first enzymes found to possess both type II terpene cyclase (TC) and prenyltransferase (PT) activities. These enzymes were characterized by heterologous expression in Aspergillus oryzae and in vitro experiments with wild-type, mutated, and truncated enzymes. Read More

    Substrate Recognition by a Dual-Function P450 Monooxygenase GfsF Involved in FD-891 Biosynthesis.
    Chembiochem 2017 Sep 4. Epub 2017 Sep 4.
    Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
    GfsF is a multifunctional P450 monooxygenase that catalyzes epoxidation and subsequent hydroxylation in the biosynthesis of macrolide polyketide FD-891. Here, we describe the biochemical and structural analysis of GfsF. To obtain the structural basis of a dual-function reaction, we determined the crystal structure of ligand-free GfsF, which revealed GfsF to have a predominantly hydrophobic substrate binding pocket. Read More

    The Oxidation of Hydrophobic Aromatic Substrates by Using a Variant of the P450 Monooxygenase CYP101B1.
    Chembiochem 2017 Sep 3. Epub 2017 Sep 3.
    Department of Chemistry, University of Adelaide, Adelaide, SA, 5005, Australia.
    The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency than norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which, in the latter enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was mutated to phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. Read More

    Ribbon-like Foldamers for Cellular Uptake and Drug Delivery.
    Chembiochem 2017 Sep 1. Epub 2017 Sep 1.
    Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Université de Montpellier, CNRS, ENSCM, 15 avenue Charles Flahault, B. P.14491, 34093, Montpellier Cedex 5, France.
    Different intracellular delivery systems of bioactive compounds have been developed, including cell-penetrating peptides. Although usually nontoxic and biocompatible, these vectors share some of the general drawbacks of peptides, notably low bioavailability and susceptibility to protease degradation, that limit their use. Herein, the conversion of short peptide sequences into poly-α-amino-γ-lactam foldamers that adopt a ribbon-like structure is investigated. Read More

    Convergent Evolution of Ergothioneine Biosynthesis in Cyanobacteria.
    Chembiochem 2017 Sep 1. Epub 2017 Sep 1.
    Department for Chemistry, University of Basel, Postfach 3350, Mattenstrasse 24a, 4002, Basel, Switzerland.
    Biosynthesis of N-α-trimethyl-2-thiohistidine (ergothioneine) is a frequent trait in cyanobacteria. This sulfur compound may provide essential relief from oxidative stress related to oxygenic photosynthesis. The central steps in ergothioneine biosynthesis are catalyzed by a histidine methyltransferase and an iron-dependent sulfoxide synthase. Read More

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