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    Characterisation of CYP102A25 from B. marmarensis and CYP102A26 from P. halophilus: P450 homologues of BM3 with preference towards hydroxylation of medium chain fatty acids.
    Chembiochem 2017 Dec 8. Epub 2017 Dec 8.
    University of Manchester, Manchester Institute of Biotechnology, 131 Princess St, M1 7DN, Manchester, UNITED KINGDOM.
    Cytochrome P450 monooxgenases are highly desired biocatalysts due to their ability to catalyse a wide variety of chemically challenging C-H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self-sufficient proteins consisting of a haem and FMN-FAD reductase domain fused in a single component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω-1, ω-2 and ω-3 hydroxy acids. Read More

    A single mutation increases the activity and stability of Pectobacterium carotovorum nitrile reductase.
    Chembiochem 2017 Dec 7. Epub 2017 Dec 7.
    State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, CHINA.
    Nitrile reductase is considered as a promising and environmentally benign nitrile-reducing biocatalyst to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of nitrile reductases reported so far are very low. To date, any attempts by protein engineering to increase the catalytic activity of nitrile reductase have failed. Read More

    Euphorbia tirucalli β-amyrin cyclase: Trp612 and Tyr736 π-electrons participate in a cation-π interaction with the transient cation and a CH-π interaction with Leu734, respectively.
    Chembiochem 2017 Dec 6. Epub 2017 Dec 6.
    Faculty of Agriculture, Niigata University, Department Applied Biological Chemsitry, Ikarashi 2-8050, Nishi-ku, 950-2181, Niigata, JAPAN.
    The functions of Trp612, Leu734 and Tyr736 of Euphorbia tirucalli β-amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5% of the wild-type activity for the W612H variant, 43% for W612F, and 63% for W612Y. Read More

    Spontaneous covalent self-assembly of the Azoarcus ribozyme from five fragments.
    Chembiochem 2017 Dec 5. Epub 2017 Dec 5.
    Portland State University, Deptm. of Chemistry, PO Box 751, 97207, Portland, UNITED STATES.
    Spontaneous covalent assembly of short RNA fragments has been proposed as a plausible prebiotically relevant pathway to a self-reproducing system. We had previously shown that the Azoarcus group I intron could self-assemble from four RNA fragments. Here, we extend this fragmentation to five RNAs that average <40 nucleotides in length. Read More

    Alphanumerical Visual Display Made of DNA Logic Gates for Drug Susceptibility Testing of Pathogens.
    Chembiochem 2017 Dec 5. Epub 2017 Dec 5.
    University of Central Florida, Chemistry Department, 41111 Libra Dr, PSB 255, 32816, Orlando, UNITED STATES.
    Molecular diagnostics of drug resistant pathogens requires analysis of point mutations in bacterial or viral genomes, which is usually performed by trained professionals and/or by a sophisticated computer algorithm. Here, we developed a DNA-based logic system that autonomously analyzes mutations in the genome of Mycobacterium tuberculosis complex (MTC) bacterial species and communicates the output to a human user as alphanumeric characters read by the naked eye. The five-gate system displays "O" ("no infection") for the absence of MTC infection; and "P" or "F" for passing or failing a drug susceptibility test, respectively. Read More

    The effects of pressure (0.1 - 200 MPa) and pH on the hydrolysis of cytosine at 373 K: implications for nucleotide stability around deep-sea black smokers.
    Chembiochem 2017 Dec 3. Epub 2017 Dec 3.
    Massey University, Institute of Fundamental Sciences, Riddet Road, 4442, Palmerston North, NEW ZEALAND.
    The relatively low chemical stability of cytosine compared to other nucleobases is a key concern in origin-of-life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil has remained unknown. We have determined by in situ NMR measurements that the half-life of cytosine at 373.15 K decreases from 18. Read More

    Protein Folding by Water-Soluble Cyclic Diselenides with Novel Oxidoreductase and Isomerase Activities.
    Chembiochem 2017 Dec 2. Epub 2017 Dec 2.
    Tokai University, Department of Chemistry, Kitakaname, 259-1292, Hiratsuka-shi, JAPAN.
    Protein disulfide isomerase (PDI) family, present in the endoplasmic reticulum (ER) of mammalian cells, catalyzes the formation and cleavage of disulfide bonds and thereby helps in protein folding. A decrease in PDI activity under ER stress conditions lead to protein misfolding, which is responsible for the progression of various human diseases, such as Alzheimers, Parkinsons, diabetes mellitus and atherosclerosis. In this paper, we report that water-soluble cyclic diselenides mimic the multifunctional activity of PDI family by facilitating oxidative folding, disulfide formation/reduction and repair of the scrambled disulfide bonds in misfolded proteins. Read More

    Pseudomonas fluorescens strain R124 encodes three different MIO-enzymes.
    Chembiochem 2017 Nov 28. Epub 2017 Nov 28.
    Budapest University of Technology and Economics, Department of Organic Chemistry and Technology, Szt. Gellert ter 4, H-1111, Budapest, HUNGARY.
    A number of class I lyase-like enzymes including aromatic ammonia-lyases and aromatic 2,3-aminomutases containing the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient limited cave encodes a histidine ammonia-lyase (HAL), a tyrosine/phenylalanine/histidine ammonia-lyase (XAL) and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of the nitrogen metabolism by gaining a PAM via horizontal gene transfer. The novel MIO-enzymes are potential biocatalysts in synthesis of enantiopure unnatural amino acids. Read More

    Unravelling the carbohydrate-binding preferences of the carbohydrate-binding modules of AMP-activated protein kinase.
    Chembiochem 2017 Nov 30. Epub 2017 Nov 30.
    University of Melbourne, Biochemistry and Molecular Biology, 30 Flemington Road, 3010, Parkville, AUSTRALIA.
    The β-subunit of AMP-activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a Carbohydrate Binding Module (CBM) that interacts with glycogen. While the β1- and β2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. β2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and we have recently shown that a Thr insertion in β2-CBM (Thr101) forms a pocket to accommodate branches. Read More

    Characterisation of a bacterial galactokinase with high activity and broad substrate tolerance for chemoenzymatic synthesis of 6-aminogalactose-1-phosphate and analogues.
    Chembiochem 2017 Nov 29. Epub 2017 Nov 29.
    The University of Manchester MIB, School of Chemistry, 131 Princess Street, M1 7DN, Manchester, UNITED KINGDOM.
    Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate active enzymes. There is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting group strategies. Read More

    Diversity of Polyketide Chains was Achieved by Deletion of the Tailoring Genes in the Biosynthesis of Ansatrienins.
    Chembiochem 2017 Nov 29. Epub 2017 Nov 29.
    Shandong University, School of Pharmaceutical Sciences, No. 44 West Wenhua Road, 250012, Jinan, CHINA.
    The ast gene cluster (GenBank accession number KF813023.1 and KP284551) was characterized to be responsible for the biosynthesis of ansatrienins in Streptomyces sp. XZQH13, which contains astC, astF1 and astF2 genes involved in the assembly of the N-cyclohexanoyl D-alanyl side chain and the hydroxylation of C-19, respectively. Read More

    Effect of site-specific peptide-tag labeling on the Biocatalytic Properties of thermoalkalophilic lipase from Geobacillus thermocatenulatus.
    Chembiochem 2017 Nov 28. Epub 2017 Nov 28.
    CSIC, Biocatalysis, marie curie 2 UAM , 28049, Madrid, SPAIN.
    Tailor-made peptides were investigated for a site-specific tag labeling of Geobacillus thermocatenulatus lipase (GTL). GTL was first genetically modified by introducing a unique cysteine on the lid-site of the enzyme, producing two variants (GTLσ-A193C and GTLσ- S196C). The chemical modification was performed by using a small library of cysteine-containing peptides. Read More

    Exploring PTDH-P450BM3 variants for the synthesis of drug metabolites.
    Chembiochem 2017 Nov 27. Epub 2017 Nov 27.
    University of Groningen, Department of Biochemistry, Nijenborgh 4, 9747 AG, Groningen, NETHERLANDS.
    The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase to enable cofactor recycling. Conditions for enzyme production were optimized and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds and a variety of products were detected. Read More

    2D Enzyme Cascade Network with Efficient Substrate Channeling by Swinging Arms.
    Chembiochem 2017 Nov 27. Epub 2017 Nov 27.
    Arizona State University, The Biodesign Institute, 1001 S. McAllister Ave, 85287-5601, Tempe, UNITED STATES.
    In living cells, compartmented or membrane-associated enzymes are often assembled into large networks for cooperatively catalyzing cascade reaction pathways essential for the cellular metabolism. Here, we report the assembly of an artificial 2D enzyme network of two cascade enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) on a wireframe DNA origami template. Swinging arms were used to facilitate the transport of redox intermediate of NAD+/NADH between enzyme pairs on the array. Read More

    Beating Bias in Directed Evolution of Proteins: Combining High-Fidelity On-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction.
    Chembiochem 2017 Nov 24. Epub 2017 Nov 24.
    Saturation mutagenesis (SM) constitutes a widely used technique in directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Here we show how this fundamental problem can be solved by applying high-fidelity solid-phase chemical gene synthesis on Silicon-chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with stereoselective formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. Read More

    Live Cell MicroRNA Imaging via MnO2 Nanosheets Mediated "DD-A" Hybridization Chain Reaction.
    Chembiochem 2017 Nov 23. Epub 2017 Nov 23.
    College of Chemistry and Chemical Engineering, Hunan University, State Key Laboratory of Chemo/Biosensing and Chemometrics, Lushan south road, 410082, Changsha, CHINA.
    Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNAs in biology and medicine. Here, we report a novel approach for live cell miRNAs imaging through biodegradable MnO2 nanosheets mediated "DD-A" FRET hybridization chain reaction (HCR). The MnO2 nanosheets can adsorb DNA hairpin probes and deliver them into live cells. Read More

    Engineered Biomolecular Recognition of RDX Using a Thermostable Alcohol Dehydrogenase as a Protein Scaffold.
    Chembiochem 2017 Nov 22. Epub 2017 Nov 22.
    Columbia University, Department of Chemical Engineering, 820 Mudd MC4721, 500 West 120th Street, 10027, New York, UNITED STATES.
    There are many biotechnology applications that would benefit from simple, stable proteins with engineered biomolecular recognition. Here we explored the hypothesis that a thermostable alcohol dehydrogenase (AdhD from P. furisosus) could be engineered to bind a small molecule instead of a cofactor or molecules involved in the catalytic transition state. Read More

    Rewarming the Primordial Soup: Revisitations and Rediscoveries in Prebiotic Chemistry.
    Chembiochem 2017 Nov 22. Epub 2017 Nov 22.
    Dipartimento di Scienze Ecologiche e Biologiche, Università della Tuscia, Via San Camillo De Lellis, 01100, Viterbo, Italy.
    A short history of primordial soup: In this essay we try to disclose some of the historical connections between the studies that have contributed to our current understanding of the emergence of catalytic RNA molecules and their components from an inanimate matter. Read More

    A small organic molecule blocks EGFR transport into the nucleus by the non-classical pathway resulting in repression of cancer invasion.
    Chembiochem 2017 Nov 20. Epub 2017 Nov 20.
    Chung-Ang University, Chemistry, 221 Heakseok-dong, Dongjak-gu, 156-756, Seoul, KOREA, REPUBLIC OF.
    In addition to the traditional epidermal growth factor receptor (EGFR) signaling pathways, nuclear EGFR has been shown to control multiple cellular functions, including cell proliferation and invasion. It has been reported that EGFR is transported into the nucleus after forming a complex with KPNA/KPNB1 or KPNB1. In this study, we have shown that EGFR can interact with both KP and KPNA, but EGF-activated EGFR mostly binds with KPNB1 through the pull-down assay. Read More

    Role of charge and hydrophobicity in liprotide formation: a molecular dynamics study with experimental constraints.
    Chembiochem 2017 Nov 20. Epub 2017 Nov 20.
    Aarhus University, iNANO, Gustav Wieds Vej 14, 8000, Aarhus C, DENMARK.
    Bovine α-lactalbumin (aLA) and oleate (OA) form a complex that has been intensively studied for its tumoricidal activity. Small-angle X-ray scattering (SAXS) has revealed a lipid core surrounded by partially unfolded protein. We call this type of complex a liprotide. Read More

    NAD+-dependent dehydrogenase PctP and PLP-dependent aminotransferase PctC catalyze the first post-glycosylation modification of sugar intermediate in pactamycin biosynthesis.
    Chembiochem 2017 Nov 17. Epub 2017 Nov 17.
    Tokyo Institute of Technology, Chemistry & Materials Science, O-okayama, Meguro-ku, 1528551, Tokyo, JAPAN.
    The unique five-membered aminocyclitol core of the antitumor antibiotic pactamycin originates from D-glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Here, we examined the timing of introduction of an amino group into certain sugar-derived intermediates using recombinant enzymes that are encoded in the pactamycin biosynthesis gene cluster. Read More

    Novel WS9326A derivatives and one novel Annimycin derivative with antimalarial activity are produced by Streptomyces asterosporus DSM 41452 and its mutant.
    Chembiochem 2017 Nov 17. Epub 2017 Nov 17.
    Albert-Ludwigs-Universit�t, Institut f. Pharmaz. Wissenschaften, Stefan-Meier-Str. 19 VF, 79104, Freiburg i. Br., GERMANY.
    In this study we report that Streptomyces asterosporus DSM 41452 is a producer of new molecules related to the non-ribosomal cyclodepsipeptide WS9326A and the polyketide Annimycin. S. asterosporus DSM 41452 is shown to produce six cyclodepsipeptides and peptides, WS9326A to G. Read More

    Immobilization of Superoxide Dismutase on Polyelectrolyte Functionalized Titania Nanosheets.
    Chembiochem 2017 Nov 16. Epub 2017 Nov 16.
    University of Geneva, Department of Inorganic and Analytical Chemistry, 30 Quai Ernest-Ansermet, 1205, Geneva, SWITZERLAND.
    Superoxide dismutase (SOD) enzyme was successfully immobilized on titania nanosheets (TNS) functionalized with poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte. The TNS-PDADMAC solid support was prepared by hydrothermal synthesis followed by self-assembled polyelectrolyte layer formation. It was found that the SOD strongly adsorbed on the oppositely charged TNS-PDADMAC through electrostatic and hydrophobic interactions. Read More

    A multi-perspective approach to solvent regulation of enzymatic activity: HMG-CoA reductase.
    Chembiochem 2017 Nov 15. Epub 2017 Nov 15.
    Fakultät für Chemie und Biochemie, Organische Chemie 1, Universitätsstraße 150, 44780, Bochum, GERMANY.
    HMG-CoA reductase was investigated in different organic co-solvents using kinetic and calorimetric measurements, molecular dynamics simulations and small-angle x-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme and the different solvent types and concentrations. In this way, we could elucidate the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents. Read More

    Production of hydroxynitrile lyase from D. tyermanii (DtHNL) in Komagataella phaffii and its immobilization as CLEA to generate a robust biocatalyst.
    Chembiochem 2017 Nov 13. Epub 2017 Nov 13.
    Austrian Centre of Industrial Biotechnology, Graz University of Technology, Institute for Molecular Biotechnology, Petersgasse 14, 8010, Graz, AUSTRIA.
    Hydroxynitrile lyase from the white rabbit's foot fern Davallia tyermanii (DtHNL) catalyzes the enantioselective synthesis of α-cyanohydrins, key building blocks for pharmaceutical and agrochemical industries. An efficient and competitive process necessitates the availability and robustness of the biocatalyst. Herein, the recombinant production of DtHNL1 in Komagataella phaffii is described, yielding approximately 900,000 U/L. Read More

    Specificity of Donor Structures for endo-β-N-Acetylglucosaminidase-Catalyzed Transglycosylation Reactions.
    Chembiochem 2017 Nov 10. Epub 2017 Nov 10.
    Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kyryu Gunma, 376-8515, Japan.
    To demonstrate the structural specificity of the glycosyl donor for the transglycosylation reaction by using endo-β-N-acetylglucosaminidase from Mucor hiemalis (endo-M), a series of tetrasaccharide oxazoline derivatives was synthesized. These derivatives correspond to the core structure of an asparagine-linked glycoprotein glycan with a β-mannose unit of a non-natural-type monosaccharide, including β-glucose, β-galactose, and β-talose in place of the β-mannose moiety. The transglycosylation activity of wildtype (WT) endo-M and two mutants, N175Q and N175A, was examined by using these tetrasaccharide donors with p-nitrophenyl N-acetylglucosaminide (GlcNAc-pNp). Read More

    Expanding the Scope of Sortase-Mediated Ligations Using Sortase Homologs.
    Chembiochem 2017 Nov 9. Epub 2017 Nov 9.
    Western Washington University, Chemistry, 516 High Street, WA, Bellingham, UNITED STATES.
    Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we have characterized the in vitro substrate preferences of eight sortase A homologs. Read More

    Improvement of the thermostability of a β-amino acid converting ω-transaminase using FoldX.
    Chembiochem 2017 Nov 9. Epub 2017 Nov 9.
    Karlsruher Institut fur Technologie, IBG-1, Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, GERMANY.
    ω-Transaminases (ω-TAs) are important biocatalysts for the synthesis of active, chiral pharmaceutical ingredients containing amino groups, such as β-amino acids which are important in peptidomimetics and as building blocks for drugs. However, the application of ω-TAs is limited by the availability and stability of enzymes with high conversion rates. One strategy for the synthesis and optical resolution of β-phenylalanine and other important aromatic β-amino acids, is the biotransformation utilizing an ω-transaminase from Variovorax paradoxus. Read More

    Synthesis and Immunological Evaluation of a Multicomponent Cancer Vaccine Candidate Containing a Long MUC1 Glycopeptide.
    Chembiochem 2017 Nov 9. Epub 2017 Nov 9.
    Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, Georgia, 30602, USA.
    A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Read More

    A FRET sensor to monitor bivalent SUMO-SIM interactions in SUMO chain binding.
    Chembiochem 2017 Nov 9. Epub 2017 Nov 9.
    Westfälische Wilhelms-Universität Münster, Institut für Biochemie, Wilhelm-Klemm-Str. 2, 48149, Münster, GERMANY.
    The small ubiquitin-like modifier (SUMO) can be assembled into polymeric chains as part of its diverse biochemical signal pattern when conjugated to substrate proteins. SUMO chain recognition is facilitated by receptor proteins containing at least two SUMO-interacting motifs (SIMs). Only little is known about the structure of SUMO chains, both in unliganded form and when complexed with multi-SIM protein partners. Read More

    Phage display on anti-infective target DXS led to an acceptor-substrate competitive peptidic inhibitor.
    Chembiochem 2017 Nov 8. Epub 2017 Nov 8.
    Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Drug Design and Optimization, Campus E8.1, 66123, Saarbrücken, GERMANY.
    Enzymes of the 2-C-methyl-d-erythritol-4-phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false positive hits. The enriched peptide binder P12 emerged as substrate (d-glyceraldehyde 3-phosphate) competitive against Deinococcus radiodurans DXS. Read More

    Enzymes as enhancers for biodegradation of synthetic polymers in wastewater.
    Chembiochem 2017 Nov 8. Epub 2017 Nov 8.
    Austrian Centre of Industrial Biotechnology, Biotechnology and Material processing, Konrad Lorenz Strasse 20, 3430, Tull an der Donau, AUSTRIA.
    Synthetic polyesters are today the second largest class of ingredient in household products and are entering WWTP after product utilization. One approach to improve polymer biodegradation in wastewater would be to complement current processes with polyester hydrolyzing enzymes and their microbial producers. In this study, hydrolysis of polyoxyethylene terephthalate polymer by hydrolases from wastewater microorganisms was investigated in in-vitro and under realistic WWTP conditions. Read More

    Flavin-N5 Covalent Intermediate in the Non-redox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme.
    Chembiochem 2017 Nov 8. Epub 2017 Nov 8.
    Virginia Polytechnic Institute and State University, Biochemistry, 360 West campus Dr, 24061, Blacksburg, UNITED STATES.
    The flavin-dependent enzyme 2-haloacrylate hydratase (2-HAH) catalyzes the conversion of 2-chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme from has been expressed in Escherichia coli, purified, and characterized. 2-HAH is monomeric in solution and contains a non-covalent, yet tightly bound, FAD. Read More

    Nucleophilic Water Capture or Proton Loss: Single Amino Acid Switch Converts δ-Cadinene Synthase into Germacradien-4-ol Synthase.
    Chembiochem 2017 Nov 8. Epub 2017 Nov 8.
    School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, UK.
    δ-Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ-cadinene and about 2 % germacradien-4-ol, which is also generated from FDP by the cyclase germacradien-4-ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site-directed mutagenesis of δ-cadinene synthase. If W279 in δ-cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. Read More

    A T7 RNA Polymerase Mutant Enhances the Yield of 5'-Thienoguanosine-Initiated RNAs.
    Chembiochem 2017 Nov 8. Epub 2017 Nov 8.
    Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, 484 West 12th Avenue, Columbus, OH, 43210, USA.
    Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50 nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class III Φ6.5 promoter. Read More

    Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism.
    Chembiochem 2017 Nov 7. Epub 2017 Nov 7.
    Faculty of Pharmaceutical Sciences, University of British Columbia, 2405 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada.
    Arginine methylation is a prevalent post-translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N-methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη-hydroxyarginine generally demonstrated substrate inhibition and had improved KM values, which evoked a possible role in inhibitor design. Read More

    Laccase-mediated grafting on biopolymers and synthetic polymers - a critical review.
    Chembiochem 2017 Nov 7. Epub 2017 Nov 7.
    Wageningen University, Laboratory of Organic Chemistry, Stippeneng 4, 6703 WE, Wageningen, NETHERLANDS.
    Laccase-mediated grafting on lignocelluloses has gained considerable attention as an environmentally benign method to covalently modify wood, paper and cork. Over the past decades this technique has been employed to also modify fibres with a polysaccharide backbone such as cellulose or chitosan to infer colouration, anti-microbial activity or anti-oxidant activity to the material. The scope of this approach has been further widened by researchers who apply mediators or high redox potential laccases and those that modify synthetic polymers and proteins. Read More

    Nested PUF proteins: extending target RNA elements for gene regulation.
    Chembiochem 2017 Nov 6. Epub 2017 Nov 6.
    Kyoto University, Institute for Chemical Research, Gokasho, 611-0011, Uji, JAPAN.
    RNA-binding proteins recognizing unique sequences within large transcriptomes serve as a powerful tool to control RNA metabolism. Pumilio and fem-3 mRNA-binding factor (PUF) proteins are considered as a good candidate for such tools because they are typically composed of eight highly homologous repeat segments and can be designed to recognize arbitrary 8-nucleotide (nt) RNA sequences. However, a specific 8-nt RNA sequence is found at multiple sites in various RNAs in the transcriptome, making it difficult to specifically target a single RNA. Read More

    Toward a Novel Drug To Target the EGF-EGFR Interaction: Design of Metabolically Stable Bicyclic Peptides.
    Chembiochem 2017 Nov 6. Epub 2017 Nov 6.
    Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028, Barcelona, Spain.
    In cancer, proliferation of malignant cells is driven by overactivation of growth-signalling mechanisms, such as the epidermal growth factor receptor (EGFR) pathway. Despite its therapeutic relevance, the EGF-EGFR interaction has remained elusive to inhibition by synthetic molecules, mostly as a result of its large size and lack of binding pockets and cavities. Designed peptides, featuring cyclic motifs and other structural constraints, have the potential to modulate such challenging protein-protein interactions (PPIs). Read More

    Resveratrol Interferes with an Early step in the Fibrillation pathway of Human lysozyme and Modulates it towards less-toxic, Off-pathway Aggregates.
    Chembiochem 2017 Nov 4. Epub 2017 Nov 4.
    Jawaharlal Nehru University, School of Biotechnology, New Mehrauli road, New Delhi,, 110067, New Delhi, INDIA.
    Effect of the red wine polyphenol, resveratrol, was investigated on the amyloid fibril formation of human lysozyme (HuL), towards elucidating the mechanism of resveratrol action and probing its role as a possible modulator of lysozyme aggregation and toxicity. Using a number of biophysical tools, resveratrol was observed to alter the fibrillation kinetics of HuL and inhibit its fibrillation by binding with weak to moderate affinity to the conformations populated at the early stages of the pathway with concomitant stabilization of these initial conformations. The marginal decrease in HuL lifetime in the presence of resveratrol by time-resolved fluorescence measurements indicated the involvement of static quenching mechanism in the interaction between HuL and resveratrol. Read More

    Enhanced Permeability and Binding Activity of Isobutylene-Grafted Peptides.
    Chembiochem 2017 Nov 3. Epub 2017 Nov 3.
    Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
    We present a new peptide-macrocyclization strategy with an isobutylene graft. The reaction is mild and proceeds rapidly and efficiently both for linear and cyclic peptides. The resulting isobutylene-grafted peptides possess improved passive membrane permeability due to the shielding of the polar backbone of the amides, as demonstrated by NMR spectroscopy and molecular dynamics simulations. Read More

    Improved Enantioselectivity of Subtilisin Carlsberg Towards Secondary Alcohols by Protein Engineering.
    Chembiochem 2017 Nov 4. Epub 2017 Nov 4.
    KTH School of Biotechnology, Industrial Biotechnology, AlbaNova University Center, 106 91, Stockholm, SWEDEN.
    Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions using secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino acid residues for mutagenesis were found by combining available literature data with molecular modeling. Read More

    Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids.
    Chembiochem 2017 Nov 2. Epub 2017 Nov 2.
    Department of Chemistry, Texas A&M University, College Station, TX, 77843, USA.
    By evolving the N-terminal domain of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) that directly interacts with tRNAPyl , a mutant clone displaying improved amber-suppression efficiency for the genetic incorporation of Nϵ -(tert-butoxycarbonyl)-l-lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of Nϵ -acetyl-l-lysine and Nϵ -(4-azidobenzoxycarbonyl)-l-δ,ϵ-dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. Read More

    Online Measurement of Oxygen-Dependent Enzyme Reaction Kinetics.
    Chembiochem 2017 Oct 31. Epub 2017 Oct 31.
    Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, 2800, Lyngby, Denmark.
    As the application of biocatalysis to complement conventional chemical and catalytic approaches continues to expand, an increasing number of reactions involve poorly water-soluble substrates. At required industrial concentrations necessary for industrial implementation, this frequently leads to heterogeneous reaction mixtures composed of multiple phases. Such systems are challenging to sample, and therefore, it is problematic to measure representative component concentrations. Read More

    Bleomycin Can Cleave an Oncogenic Noncoding RNA.
    Chembiochem 2017 Oct 30. Epub 2017 Oct 30.
    Department of Chemistry, The Scripps Research Institute, 110 Scripps Way, Jupiter, FL, 33458, USA.
    Noncoding RNAs are pervasive in cells and contribute to diseases such as cancer. A question in biomedical research is whether noncoding RNAs are targets of medicines. Bleomycin is a natural product that cleaves DNA; however, it is known to cleave RNA in vitro. Read More

    Exploring the Substrate Scope of Baeyer-Villiger Monooxygenases with Branched Lactones as Entry towards Polyesters.
    Chembiochem 2017 Oct 27. Epub 2017 Oct 27.
    Maastricht University, Aachen-Maastricht Institute for Biobased Materials (AMIBM), Brightlands Chemelot Campus, Urmonderbaan 22, 6167 RD, Geleen, The Netherlands.
    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that are able to convert cyclic ketones into lactones by the insertion of oxygen. The aim of this study was to explore the substrate scope of several BVMOs with (biobased) cyclic ketones as precursors for the synthesis of branched polyesters. The product structure and the degree of conversion of several biotransformations were determined after conversions by using self-sufficient BVMOs. Read More

    Chemical approach to biological safety - Molecular level control of an integrated zinc finger nuclease.
    Chembiochem 2017 Oct 27. Epub 2017 Oct 27.
    University of Szeged, Department of Inorganic and Analyticial Chemistry, Dom ter 7, 6720, Szeged, HUNGARY.
    Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity, related to off-target cleavages. This problem could be targeted by the regulation of the nuclease domain. Here we provide the experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. Read More

    Ultrasensitive DNAzyme-Based Ca2+ Detection Boosted by Ethanol and a Solvent-Compatible Scaffold for Aptazyme Design.
    Chembiochem 2017 Oct 27. Epub 2017 Oct 27.
    Department of Chemistry and Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada.
    Functional DNA includes aptamers and DNAzymes, and metal ions are often important for achieving the chemical functions of such DNA. Biosensors based on functional DNA have mainly been tested in aqueous buffers. By introducing organic solvents with much lower dielectric constants, the interaction between metal ions and DNA can be significantly enhanced, and this might affect the performance of DNA-based biosensors. Read More

    Propensity for cis-Proline Formation in Unfolded Proteins.
    Chembiochem 2017 Oct 24. Epub 2017 Oct 24.
    Laboratory of Chemical Physics, National Institutes of Health, 5 Memorial Drive, Bethesda, MD, 20892, USA.
    In unfolded proteins, peptide bonds involving Pro residues exist in equilibrium between the minor cis and major trans conformations. Folded proteins predominantly contain trans-Pro bonds, and slow cis-trans Pro isomerization in the unfolded state is often found to be a rate-limiting step in protein folding. Moreover, kinases and phosphatases that act upon Ser/Thr-Pro motifs exhibit preferential recognition of either the cis- or trans-Pro conformer. Read More

    Characterization of Terminators in Saccharomyces cerevisiae and an Exploration of Factors Affecting Their Strength.
    Chembiochem 2017 Oct 23. Epub 2017 Oct 23.
    School of Chemistry and Chemical Engineering, Key Laboratory for Green Processing of Chemical, Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi, 832003, P.R. China.
    Terminators in eukaryotes play an important role in regulating the transcription process by influencing mRNA stability, translational efficiency, and localization. Herein, the strengths of 100 natural terminators in Saccharomyces cerevisiae have been characterized by inserting each terminator downstream of the TYS1p-enhanced green fluorescent protein (eGFP) reporter gene and measuring the fluorescent intensity (FI) of eGFP. Within this library, there are 45 strong terminators, 31 moderate terminators, and 24 weak terminators. Read More

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