ChemBioChem

Surface Chemistry and Cell Biological Tools for the Analysis of Cell Adhesion and Migration.

Publication Date: 2010 Mar 2 PMID: 20198673
Authors: Pulsipher, A. - Yousaf, M. N.
Journal: Chembiochem

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Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions.

Publication Date: 2010 Feb 28 PMID: 20191656
Authors: Marsh, E. N. - Patterson, D. P. - Li, L.
Journal: Chembiochem

Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors: ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B(12), AdoCbl) or by single-electron reduction of S-adenosylmethionine (AdoMet) complexed to an iron-sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine's role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen, which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations; nevertheless, mechanistic insights gained from studying these enzymes have proved extremely valuable in understanding how enzymes generate and control highly reactive free radical intermediates. In contrast, there has been a recent explosion in the number of radical-AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions; here there is much still to learn about their mechanisms. Although all the radical-AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.

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Perspectives on Synthetic Promoters for Biocatalysis and Biotransformation.

Publication Date: 2010 Feb 28 PMID: 20191652
Authors: Ruth, C. - Glieder, A.
Journal: Chembiochem

Acting on the transcriptional level, synthetic promoters have been useful tools for controlling gene expression and have applications in many fields. Here, we discuss synthetic promoters and libraries in regard to current and future applications in the field of biocatalysis or biotransformation. We also focus on synthetic promoter design principles and distinguish between prokaryotic and eukaryotic destinations. The natural toolboxes available for tuneable gene expression and the regulation of enzyme function are limited and primarily host specific. Synthetic biology offers generally applicable concepts and quick implementation. Smart alternatives to transcriptional regulation enrich the engineer's tool box for optimizing industrial enzyme production and host-cell physiology for whole-cell processes. Industrially applicable, tuneable enzyme cascades and artificial circuits for iterative up- and down-regulation will soon be achieved.

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Zinc Fingered: New Compounds that Thwart Gram-Positive Biofilm Formation by Sequestering Zinc.

Publication Date: 2010 Feb 28 PMID: 20191651
Authors: Musk, D. J. Jr
Journal: Chembiochem

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Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One- and Two-Photon Illumination.

Publication Date: 2010 Feb 24 PMID: 20187057
Authors: Kumar Sinha, D. - Neveu, P. - Gagey, N. - Aujard, I. - Benbrahim-Bouzidi, C. - Le Saux, T. - Rampon, C. - Gauron, C. - Goetz, B. - Dubruille, S. - Baaden, M. - Volovitch, M. - Bensimon, D. - Vriz, S. - Jullien, L.
Journal: Chembiochem

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.

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An Expeditious Route to Fluorinated Rapamycin Analogues by Utilising Mutasynthesis.

Publication Date: 2010 Feb 23 PMID: 20186904
Authors: Goss, R. J. - Lanceron, S. - Deb Roy, A. - Sprague, S. - Nur-E-Alam, M. - Hughes, D. L. - Wilkinson, B. - Moss, S. J.
Journal: Chembiochem

Rapamycin is a drug with several important clinical uses. Its complex structure means that total synthesis of this natural product and its analogues is demanding and lengthy. A more expeditious approach is to utilise biosynthesis to enable the generation of otherwise synthetically intractable analogues. In order to achieve this, rules governing biosynthetic precursor substrate preference must be established. Through determining these rules and synthesising and administering suitable substrate precursors, we demonstrate the first generation of fluorinated rapamycin analogues. Here we report the generation of six new fluororapamycins.

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A Step Closer to Complete Chemical Reprogramming for Generating iPS Cells.

Publication Date: 2010 Feb 22 PMID: 20183843
Authors: Solanki, A. - Lee, K. B.
Journal: Chembiochem

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2-(1-Ethynylpyrene)-Adenosine as a Folding Probe for RNA-Pyrene in or out.

Publication Date: 2010 Feb 22 PMID: 20183842
Authors: Forster, U. - Lommel, K. - Sauter, D. - Grunewald, C. - Engels, J. W. - Wachtveitl, J.
Journal: Chembiochem

A series of short RNA duplexes containing one or two 1-ethynylpyrene-modified adenine bases was synthesised. The melting behaviour of these duplexes was examined by monitoring temperature-dependent pyrene fluorescence. In the singly modified RNA duplexes, the bases flanking the ethynylpyrene-rA were varied to examine the sequence specificity of the fluorescence change of pyrene upon RNA hybridisation. Because an increase in pyrene fluorescence upon melting of the duplex can be correlated with intercalation of pyrene, and a decrease is usually associated with the position of pyrene outside the strand, a relationship between the flanking bases and the tendency of the dye to intercalate has been established. It was found that pyrene intercalation is less likely to take place if the modified base is flanked only by A-U base pairs. Flanking G-C base pairs, even only in the 5'-direction of the modified base, will favour intercalation. In addition, we examined a doubly modified compound that had a pyrene located on each strand. The spectra indicated that the two pyrenes were close enough for interaction. Upon melting of the strand, a fluorescence blue shift corresponding to the dissociation of the pyrene-pyrene complex could be observed in addition to the intensity effect already known from the singly modified compounds. Two melting curves based on the different properties of the fluorophore could be extracted, leading to different melting points corresponding to the global duplex melting and to the change of local pyrene environment, respectively.

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Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2.

Publication Date: 2010 Feb 22 PMID: 20183841
Authors: Zehentgruber, D. - Hannemann, F. - Bleif, S. - Bernhardt, R. - Lutz, S.
Journal: Chembiochem

Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C--H bonds mainly in a stereo- and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2-catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18-fold to yield an absolute productivity up to 5.5 g L(-1) d(-1). Product extraction with absorber resins allowed the recovery of quantitative amounts of 15beta-OH-progesterone and 15beta-OH-testosterone and also the reuse of the biocatalyst.

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Noncovalent-Interaction-Promoted Ligation for Protein Labeling.

Publication Date: 2010 Feb 19 PMID: 20175182
Authors: Hori, Y. - Egashira, Y. - Kamiura, R. - Kikuchi, K.
Journal: Chembiochem

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Preview: ChemBioChem 5/2010.

Publication Date: 2010 Mar 1 PMID: 20175075
Authors:
Journal: Chembiochem

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Spotlights on our sister journals: ChemBioChem 4/2010.

Publication Date: 2010 Mar 1 PMID: 20175074
Authors:
Journal: Chembiochem

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Inside Cover: Unnatural Polyketide Analogues Selectively Target the HER Signaling Pathway in Human Breast Cancer Cells (ChemBioChem 4/2010).

Publication Date: 2010 Mar 1 PMID: 20175073
Authors: Kwon, S. J. - Kim, M. I. - Ku, B. - Coulombel, L. - Kim, J. H. - Shawky, J. H. - Linhardt, R. J. - Dordick, J. S.
Journal: Chembiochem

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Turning Riboswitches Loose.

Publication Date: 2010 Feb 16 PMID: 20162658
Authors: Hartig, J. S.
Journal: Chembiochem

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Chemical protein synthesis by kinetically controlled ligation of Peptide o-esters.

Publication Date: 2010 Mar 1 PMID: 20157912
Authors: Zheng, J. S. - Cui, H. K. - Fang, G. M. - Xi, W. X. - Liu, L.
Journal: Chembiochem

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Sugar-Selective Enrichment of a D-Glucose-Substituted Ruthenium Bipyridyl Complex Inside HepG2 Cancer Cells.

Publication Date: 2010 Feb 15 PMID: 20157911
Authors: Gottschaldt, M. - Schubert, U. S. - Rau, S. - Yano, S. - Vos, J. G. - Kroll, T. - Clement, J. - Hilger, I.
Journal: Chembiochem

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Directed Evolution of an Antitumor Drug (Arginine Deiminase PpADI) for Increased Activity at Physiological pH.

Publication Date: 2010 Feb 15 PMID: 20157910
Authors: Zhu, L. - Tee, K. L. - Roccatano, D. - Sonmez, B. - Ni, Y. - Sun, Z. H. - Schwaneberg, U.
Journal: Chembiochem

Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH-dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed-evolution protocol based on an adapted citrulline-screening protocol in microtiter-plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild-type PpADI and retains ~50 % of its activity relative to its pH optimum, compared to ~10 % in the case of the wild-type PpADI.

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Redesigning the Active Site of Transaldolase TalB from Escherichia coli: New Variants with Improved Affinity towards Nonphosphorylated Substrates.

Publication Date: 2010 Feb 10 PMID: 20148428
Authors: Schneider, S. - Gutierrez, M. - Sandalova, T. - Schneider, G. - Clapes, P. - Sprenger, G. A. - Samland, A. K.
Journal: Chembiochem

Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X-ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site-directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D-fructose from DHA and d,l-GA by using an adjusted colour assay. The best results with respect to the synthesis of D-fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l-GA (K(M)=24 mM). We demonstrated that this double mutant can use D-GA, glycolaldehyde and the L-isomer, L-GA, as acceptor substrates. This resulted in preparative synthesis of D-fructose, D-xylulose and L-sorbose when DHA was used as donor. Hence, we engineered a DHA-dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.

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Click-Chemistry-Derived Tetracycline-Amino Acid Conjugates Exhibiting Exceptional Potency and Exclusive Recognition of the Reverse Tet Repressor.

Publication Date: 2010 Feb 10 PMID: 20148427
Authors: Usai, I. - Krueger, M. - Einsiedel, J. - Hillen, W. - Gmeiner, P.
Journal: Chembiochem

A click-chemistry-based synthesis of biologically active doxycycline-amino acid conjugates is described. Starting from 9-aminodoxycycline derivatives and complementary functionalized amino acids, ligation was accomplished by copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC). The final products were tested in a variety of TetR and revTetR systems, and the C-terminally linked phenylalanine conjugate 12 c exhibited high selectivity for revTetR over TetR. Besides the unique property of the specific effector 12 c to effectively differentiate TetR and its reverse phenotype, the test compound proved to be almost devoid of any antibacterial activity; this will be highly beneficial for future applications to control gene expression in bacterial systems.

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Biological Iron-Monosulfide Production for Efficient Electricity Harvesting from a Deep-Sea Metal-Reducing Bacterium.

Publication Date: 2010 Feb 9 PMID: 20146276
Authors: Nakamura, R. - Okamoto, A. - Tajima, N. - Newton, G. J. - Kai, F. - Takashima, T. - Hashimoto, K.
Journal: Chembiochem

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Cloning and characterization of the ravidomycin and chrysomycin biosynthetic gene clusters.

Publication Date: 2010 Mar 1 PMID: 20140934
Authors: Kharel, M. K. - Nybo, S. E. - Shepherd, M. D. - Rohr, J.
Journal: Chembiochem

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1-1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D-ravidosamine and D-virenose biosynthetic genes, post-PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross-complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino- and neutral sugars, exemplified through the structurally distinct 6-membered D-ravidosamine and 5-membered D-fucofuranose, to the coumarin-based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.

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Synthetic chain terminators off-load intermediates from a type I polyketide synthase.

Publication Date: 2010 Mar 1 PMID: 20135665
Authors: Tosin, M. - Betancor, L. - Stephens, E. - Ariel Li, W. M. - Spencer, J. B. - Leadlay, P. F.
Journal: Chembiochem

Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily-analysed form. We prepared a series of nonhydrolysable pantetheine and N-acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off-load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6-deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC-HR-ESI-MS, are the first of their kind and prove that small-molecule chain terminators can be used as convenient probes of the biosynthetic process.

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Chemical RNA modifications for studies of RNA structure and dynamics.

Publication Date: 2010 Mar 1 PMID: 20135663
Authors: Wachowius, F. - Hobartner, C.
Journal: Chembiochem

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Peering into the black box of fungal polyketide biosynthesis.

Publication Date: 2010 Mar 1 PMID: 20127928
Authors: Weissman, K. J.
Journal: Chembiochem

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Identification of the Tirandamycin Biosynthetic Gene Cluster from Streptomyces sp. 307-9.

Publication Date: 2010 Mar 1 PMID: 20127927
Authors: Carlson, J. C. - Fortman, J. L. - Anzai, Y. - Li, S. - Burr, D. A. - Sherman, D. H.
Journal: Chembiochem

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl-tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307-9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase-nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly-line oxidative modifications.

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Chemically tailored multivalent virus platforms: from drug delivery to catalysis.

Publication Date: 2010 Mar 1 PMID: 20127782
Authors: Udit, A. K. - Hackenberger, C. P. - O'Reilly, M. K.
Journal: Chembiochem

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In vivo Production of Functional Single-Chain Fv Fragment with an N-Terminal-Specific Bio-orthogonal Reactive Group.

Publication Date: 2010 Mar 1 PMID: 20127780
Authors: Selvakumar, E. - Rameshkumar, N. - Lee, S. G. - Lee, S. J. - Park, H. S.
Journal: Chembiochem

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Peptide-functionalized spherical polyelectrolyte nanobrushes for real-time sensing of protease activity.

Publication Date: 2010 Mar 1 PMID: 20112322
Authors: Yin, B. C. - Zhang, M. - Tan, W. - Ye, B. C.
Journal: Chembiochem

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Enzymatic thioxyloside synthesis: characterization of thioglycoligase variants identified from a site-saturation mutagenesis library of bacillus circulans xylanase.

Publication Date: 2010 Mar 1 PMID: 20112321
Authors: Armstrong, Z. - Reitinger, S. - Kantner, T. - Withers, S. G.
Journal: Chembiochem

Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site-saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.

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Enzyme Catalytic Promiscuity: The Nonheme Fe(2+) Center of beta-Diketone-Cleaving Dioxygenase Dke1 Promotes Hydrolysis of Activated Esters.

Publication Date: 2010 Mar 1 PMID: 20112320
Authors: Leitgeb, S. - Nidetzky, B.
Journal: Chembiochem

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FACS-Based Selection of Tandem Tetracysteine Peptides with Improved ReAsH Brightness in Live Cells.

Publication Date: 2010 Mar 1 PMID: 20099291
Authors: Van Engelenburg, S. B. - Nahreini, T. - Palmer, A. E.
Journal: Chembiochem

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A MAS NMR Study of the Bacterial ABC Transporter ArtMP.

Publication Date: 2010 Mar 1 PMID: 20099290
Authors: Lange, V. - Becker-Baldus, J. - Kunert, B. - van Rossum, B. J. - Casagrande, F. - Engel, A. - Roske, Y. - Scheffel, F. M. - Schneider, E. - Oschkinat, H.
Journal: Chembiochem

ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively (13)C,(15)N-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment.

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Monitoring the activity of 2-oxoglutarate dependent histone demethylases by NMR spectroscopy: direct observation of formaldehyde.

Publication Date: 2010 Mar 1 PMID: 20095001
Authors: Hopkinson, R. J. - Hamed, R. B. - Rose, N. R. - Claridge, T. D. - Schofield, C. J.
Journal: Chembiochem

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Structural Diversity of PDZ-Lipid Interactions.

Publication Date: 2010 Mar 1 PMID: 20091728
Authors: Gallardo, R. - Ivarsson, Y. - Schymkowitz, J. - Rousseau, F. - Zimmermann, P.
Journal: Chembiochem

PDZ domains are globular protein modules that are over-and-above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ-phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin-1, syntenin-2, PTP-Bas, PAR-3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ-lipid interactions and the consequences for peptide interactions.

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Molecular evolution of fern squalene cyclases.

Publication Date: 2010 Feb 15 PMID: 20082400
Authors: Shinozaki, J. - Shibuya, M. - Takahata, Y. - Masuda, K. - Ebizuka, Y.
Journal: Chembiochem

Triterpenes, a diverse group of natural products comprising six isoprene units, are distributed across various organisms from bacteria to higher plants. Ferns are sporophytes that produce triterpenes and are lower on the evolutionary scale than higher plants. Among ferns that produce triterpenes analogous to bacterial hopanoids, Polypodiodes niponica produces migrated dammaranes and oleananes, which are also widely found in higher plants. Because the study of terpene-producing ferns could help us to understand the molecular basis of triterpene biosynthesis, cDNA cloning of squalene cyclases (SCs) from P. niponica was carried out. Two SCs (PNT and PNG) were obtained. The heterologously expressed PNT produces tirucalla-7,21-diene (67% major), and PNG produces germanicene (69%). Phylogenetic analysis revealed that PNT and PNG, which produce higher-plant-type migrated dammaranes and oleananes, are closely related to bacterial-type SCs. Furthermore, analysis of the minor products indicated that fern SCs gained the ability to directly form dammarenyl cations, which are key intermediates in oleanane formation during molecular evolution.

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Identification and characterization of a peptidic ligand for ras.

Publication Date: 2010 Mar 1 PMID: 20082398
Authors: Gareiss, P. C. - Schneekloth, A. R. - Salcius, M. J. - Seo, S. Y. - Crews, C. M.
Journal: Chembiochem

The development of new ligands for the oncoprotein Ras can provide tools for the study of this important signaling component or potentially serve as therapeutic agents for the treatment of Ras-associated diseases. Herein, we report a peptidic Ras ligand identified through naive phage display. Panning a phage library with a diversity of 10(9) transormants successfully identified a peptide dodecamer that contains two internal consensus motifs and binds Ras in both the active GTP- and inactive GDP-bound conformations with low micromolar dissociation constants. The dodecamer does not alter the intrinsic GTPase activity of Ras, does not compete for Ras binding to the Ras binding domain of Raf, and does not alter cell viability. This novel Ras ligand has the potential to serve in the development of higher-affinity ligands and chemical tools targeting Ras.

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Turning the 10-23 DNAzyme on and off with light.

Publication Date: 2010 Feb 15 PMID: 20077457
Authors: Richards, J. L. - Seward, G. K. - Wang, Y. H. - Dmochowski, I. J.
Journal: Chembiochem

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Architectural repertoire of ligand-binding pockets on protein surfaces.

Publication Date: 2010 Mar 1 PMID: 20069621
Authors: Weisel, M. - Kriegl, J. M. - Schneider, G.
Journal: Chembiochem

Knowledge of the three-dimensional structure of ligand binding sites in proteins provides valuable information for computer-assisted drug design. We present a method for the automated extraction and classification of ligand binding site topologies, in which protein surface cavities are represented as branched frameworks. The procedure employs a growing neural gas approach for pocket topology assignment and pocket framework generation. We assessed the structural diversity of 623 known ligand binding site topologies based on framework cluster analysis. At a resolution of 5 A only 23 structurally distinct topology groups were formed; this suggests an overall limited structural diversity of ligand-accommodating protein cavities. Higher resolution allowed for identification of protein-family specific pocket features. Pocket frameworks highlight potentially preferred modes of ligand-receptor interactions and will help facilitate the identification of druggable subpockets suitable for ligand affinity and selectivity optimization.

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Reversible light switching of cell signalling by genetically encoded protein dimerization.

Publication Date: 2010 Feb 15 PMID: 20063337
Authors: Georgianna, W. E. - Deiters, A.
Journal: Chembiochem

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The role of flexibility in the rational design of modularly assembled ligands targeting the RNAs that cause the myotonic dystrophies.

Publication Date: 2010 Feb 15 PMID: 20058255
Authors: Disney, M. D. - Lee, M. M. - Pushechnikov, A. - Childs-Disney, J. L.
Journal: Chembiochem

Modularly assembled ligands were designed to target the RNAs that cause two currently untreatable neuromuscular disorders, myotonic dystrophy types 1 (DM1) and 2 (DM2). DM1 is caused by an expanded repeating sequence of CUG, and DM2 is caused by expanded CCUG repeats. Both are present in noncoding regions and fold into hairpins with either repeating 1x1 nucleotide UU (DM1) or 2x2 nucleotide 5'-CU/3'-UC (DM2) internal loops separated by two GC pairs. The repeats are toxic because they sequester the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Rational design of ligands targeting these RNAs was enabled by a database of RNA motif-ligand partners compiled by using two-dimensional combinatorial screening (2DCS). One 2DCS study found that the 6''-azido-kanamycin A module binds internal loops similar to those found in DM1 and DM2. In order to further enhance affinity and specificity, the ligand was assembled on a peptoid backbone to precisely control valency and the distance between ligand modules. Designed compounds are more potent and specific binders to the toxic RNAs than MBNL1 and inhibit the formation of the RNA-protein complexes with nanomolar IC(50) values. This study shows that three important factors govern potent inhibition: 1) the surface area sequestered by the assembled ligands; 2) the spacing between ligand modules since a longer distance is required to target DM2 RNAs than DM1 RNAs; and 3) flexibility in the modular assembly scaffold used to display the RNA-binding module. These results have impacts on the general design of assembled ligands targeting RNAs present in genomic sequence.

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Lower homologues of okoumal and disila-okoumal: synthesis and olfactory characterization of novel ambergris odorants.

Publication Date: 2010 Feb 15 PMID: 20058254
Authors: Natscher, J. B. - Laskowski, N. - Kraft, P. - Tacke, R.
Journal: Chembiochem

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Unnatural Polyketide Analogues Selectively Target the HER Signaling Pathway in Human Breast Cancer Cells.

Publication Date: 2010 Mar 1 PMID: 20058253
Authors: Kwon, S. J. - Kim, M. I. - Ku, B. - Coulombel, L. - Kim, J. H. - Shawky, J. H. - Linhardt, R. J. - Dordick, J. S.
Journal: Chembiochem

Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets. Several plant polyphenols including the type III polyketide synthase products (genistein, curcumin, resveratrol, and epigallocatechin-3-galate) possess chemopreventive activity, primarily as a result of RTK inhibition. However, only a small fraction of the polyphenolic structural universe has been evaluated. Along these lines, we have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogues with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme A (CoA) and methylmalonyl-CoA as extender substrates. The resulting polyketide analogues possessed a similar structural polyketide backbone (aromatic-2-pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT-474 cells resulted in identification of several trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of nontransformed MCF-10A breast cells (IC(50)>100 muM). Specifically, 4-trifluoromethylcinnamoyl pyrone (compound 2 e) was highly potent (IC(50)<200 nM) among the test compounds toward proliferation of several breast cancer cell lines. This breadth of activity likely stems from the ability of compound 2 e to inhibit the phosphorylation of HER1, HER2, and HER3. Therefore, these polyketide analogues might prove to be useful drug candidates for potential breast cancer therapy.

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Blue fluorescent amino acids as in vivo building blocks for proteins.

Publication Date: 2010 Feb 15 PMID: 20058252
Authors: Merkel, L. - Hoesl, M. G. - Albrecht, M. - Schmidt, A. - Budisa, N.
Journal: Chembiochem

In vivo expression of colored proteins without post-translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell-permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red-shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2-, 4-, 5-, 6-, and 7-azaindole) 4-azaindole has exhibited the largest Stokes shift (approximately 130 nm) in steady-state fluorescence measurements. It is also highly biocompatible and as 4-azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono- and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4-azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole-containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo- or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady-state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.

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Real-time monitoring of RNA synthesis in a phospholipid-coated microdroplet as a live-cell model.

Publication Date: 2010 Feb 15 PMID: 20058251
Authors: Tsuji, A. - Yoshikawa, K.
Journal: Chembiochem

We constructed a phospholipid-coated water-in-oil microdroplet (20-60 microm in diameter) that encapsulated plasmid DNA containing a human beta-actin cDNA sequence, fluorescent oligonucleotide probes, and other components of the transcription reaction. Transcription inside individual microdroplets was investigated in real time by using fluorescence microscopy. The progress of the transcription reaction was successively monitored by fluorescence resonance energy transfer (FRET), which was derived from the specific hybridization of fluorescent oligonucleotide probes to the beta-actin mRNAs synthesized. In microdroplets composed of phosphatidylethanolamine, DNAs were located in the aqueous phase or on the membrane surface depending on the Mg(II) concentration. FRET images showed that transcription occurred on DNA in both states. Moreover, individual DNA molecules undergoing transcription were visualized as discrete FRET signals in cases in which small numbers of DNAs were present in the microdroplet.

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5-Stabilized phosphatidylinositol 3,4,5-trisphosphate analogues bind Grp1 PH, inhibit phosphoinositide phosphatases, and block neutrophil migration.

Publication Date: 2010 Feb 15 PMID: 20052709
Authors: Zhang, H. - He, J. - Kutateladze, T. G. - Sakai, T. - Sasaki, T. - Markadieu, N. - Erneux, C. - Prestwich, G. D.
Journal: Chembiochem

Metabolically stabilized analogues of PtdIns(3,4,5)P3 have shown long-lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5-phosphatase-resistant analogues of PtdIns(3,4,5)P3, the 5-methylene phosphonate (MP) and 5-phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P3 analogues in four contexts. First, the relative binding affinities of the 3-MP, 3-PT, 5-MP, 5-PT, and 3,4,5-PT3 analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P3. Third, exogenously delivered analogues severely impair complement factor C5a-mediated polarization and migration of murine neutrophils. Finally, the new analogues show long-lived agonist activity in mimicking insulin action in sodium transport in A6 cells.

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HER2- and EGFR-specific affiprobes: novel recombinant optical probes for cell imaging.

Publication Date: 2010 Feb 15 PMID: 20052708
Authors: Lyakhov, I. - Zielinski, R. - Kuban, M. - Kramer-Marek, G. - Fisher, R. - Chertov, O. - Bindu, L. - Capala, J.
Journal: Chembiochem

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell-surface receptors/tyrosine kinases. EGFR- and HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683-696). We have created photostable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/ receptors.

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Protection of human genomic DNA from mechanical stress by reversible folding transition.

Publication Date: 2010 Feb 15 PMID: 20049761
Authors: Cinque, L. - Ghomchi, Y. - Chen, Y. - Bensimon, A. - Baigl, D.
Journal: Chembiochem

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Glyco-scan: varying glycosylation in the sequence of the peptide hormone PYY3-36 and its effect on receptor selectivity.

Publication Date: 2010 Feb 15 PMID: 20049760
Authors: Pedersen, S. L. - Steentoft, C. - Vrang, N. - Jensen, K. J.
Journal: Chembiochem

The increasing prevalence of obesity worldwide calls for safe and highly efficacious satiety drugs. PYY3-36 has been implicated in food intake regulation, and novel peptide analogues with high Y2 receptor-subtype selectivity and potency have potential as drugs for the treatment of obesity. It has been hypothesized that PYY3-36 associates with the plasma membrane prior to receptor activation such that the amphipathic alpha-helix of PYY3-36 possibly guides the C-terminal pentapeptide into the correct conformation for receptor activation. Ala-scans are used routinely to study the effect of individual amino acids in a given peptide sequence. Here we report the glyco-scan of the peptide hormone PYY3-36, in which hydroxyl side-chain functionalities were glycosylated; in addition new glycosylation sites were introduced. An array of novel PYY3-36 analogues with a glycan positioned in the water-membrane interface or in the N terminal were screened for Y-receptor affinity and selectivity as well as metabolic stability. Interestingly, in contrast to the Y1 and Y4 receptors, the Y2 receptor readily accommodated glycosylations. Especially glycosylations in the alpha-helical region of PYY3-36 were favorable both in terms of Y-receptor selectivity and endopeptidase resistance. We thus report several PYY3-36 analogues with enhanced Y-receptor selectivity. Our results can be used in the design of novel PYY analogues for the treatment of obesity. The glyco-scan concept, as systematically demonstrated here, has the potential for a wider applicability.

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Mutant lipase-catalyzed kinetic resolution of bulky phenyl alkyl sec-alcohols: a thermodynamic analysis of enantioselectivity.

Publication Date: 2010 Feb 15 PMID: 20049759
Authors: Vallin, M. - Syren, P. O. - Hult, K.
Journal: Chembiochem

The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.

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Chemical synthesis and biological screening of 2-aminoimidazole-based bacterial and fungal antibiofilm agents.

Publication Date: 2010 Feb 15 PMID: 20049758
Authors: Rogers, S. A. - Bero, J. D. - Melander, C.
Journal: Chembiochem

A collection of 2-aminoimidazole/triazole amides has been synthesized and screened for antibiofilm activity. This class of small molecules was found to modulate the biofilm activity of Pseudomonas aeruginosa, a multidrug-resistant strain of Acinetobacter baumannii (MDRAB), a methicillin-resistant Staphylococcus aureus strain (MRSA), Escherichia coli, Rhodospirillum salexigens, Staphylococcus epidermidis, Vibrio vulnificus, and vancomycin-resistant Enterococcus faecium as well as the yeast Candida albicans and Cryptococcus neoformans. Furthermore, lead compounds were found to not lyse red blood cells at active concentrations.

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Jewels in the pearl.

Publication Date: 2010 Feb 15 PMID: 20043309
Authors: Weiss, I. M.
Journal: Chembiochem

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Pathways and substrate specificity of DMSP catabolism in marine bacteria of the Roseobacter clade.

Publication Date: 2010 Feb 15 PMID: 20043308
Authors: Dickschat, J. S. - Zell, C. - Brock, N. L.
Journal: Chembiochem

The volatiles released by Phaeobacter gallaeciensis, Oceanibulbus indolifex and Dinoroseobacter shibae have been investigated by GC-MS, and several MeSH-derived sulfur volatiles have been identified. An important sulfur source in the oceans is the algal metabolite dimethylsulfoniopropionate (DMSP). Labelled [2H6]DMSP was fed to the bacteria to investigate the production of volatiles from this compound through the lysis pathway to [2H6]dimethylsulfide or the demethylation pathway to [2H3]-3-(methylmercapto)propionic acid and lysis to [2H3]MeSH. [2H6]DMSP was efficiently converted to [2H3]MeSH by all three species. Several DMSP derivatives were synthesised and used in feeding experiments. Strong dealkylation activity was observed for the methylated ethyl methyl sulfoniopropionate and dimethylseleniopropionate, as indicated by the formation of EtSH- and MeSeH-derived volatiles, whereas no volatiles were formed from dimethyltelluriopropionate. In contrast, the dealkylation activity for diethylsulfoniopropionate was strongly reduced, resulting in only small amounts of EtSH-derived volatiles accompanied by diethyl sulfide in P. gallaeciensis and O. indolifex, while D. shibae produced the related oxidation product diethyl sulfone. The formation of diethyl sulfide and diethyl sulfone requires the lysis pathway, which is not active for [2H6]DMSP. These observations can be explained by a shifted distribution between the two competing pathways due to a blocked dealkylation of ethylated substrates.

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A beta/gamma motif to mimic alpha-helical turns in proteins.

Publication Date: 2010 Feb 15 PMID: 20039254
Authors: Rezaei Araghi, R. - Jackel, C. - Colfen, H. - Salwiczek, M. - Volkel, A. - Wagner, S. C. - Wieczorek, S. - Baldauf, C. - Koksch, B.
Journal: Chembiochem

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Uracil-catalyzed synthesis of acetyl phosphate: a photochemical driver for protometabolism.

Publication Date: 2010 Feb 15 PMID: 20039252
Authors: Hagan, W. J. Jr
Journal: Chembiochem

Progress toward a protometabolism (the earliest energy storage networks) has been severely hindered by a shortage of driver reactions, which could have harnessed solar photons or coupled electron sources/sinks on the primordial Earth. Here, it is reported for the first time that thioacetate can be converted into a known metabolite, acetyl phosphate, by ultraviolet light and in aqueous solution at neutral pH. Of more compelling importance, the synthesis is catalyzed by uracil, which suggests that a genetic component may have also facilitated the emergence of metabolic pathways. The chemistry of acetyl phosphate has been extensively studied, and it is known to be a precursor of phosphate esters, pyrophosphate and possibly longer inorganic chains. Moreover, its bifunctional reactivity (as either an acetyl or phosphoryl donor) would have been integral for the first metabolic cycles.

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Delivery of macromolecules into live cells by simple co-incubation with a peptide.

Publication Date: 2010 Feb 15 PMID: 20029930
Authors: Lee, Y. J. - Erazo-Oliveras, A. - Pellois, J. P.
Journal: Chembiochem

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Structure and binding of peptide-dendrimer ligands to vitamin B12.

Publication Date: 2010 Feb 15 PMID: 20014271
Authors: Uhlich, N. A. - Natalello, A. - Kadam, R. U. - Doglia, S. M. - Reymond, J. L. - Darbre, T.
Journal: Chembiochem

The third-generation peptide-dendrimer B1 (AcES)8(BEA)4(K-Amb-Y)2BCD-NH2 (B=branching (S)-2,3-diaminopropanoic acid, K=branching lysine, Amb=4-aminomethyl-benzoic acid) is the first synthetic model for cobalamin-binding proteins and binds cobalamin strongly (K(a)=5.0 x 10(6) M(-1)) and rapidly (k(2)=346 M(-1) s(-1)) by coordination of cobalt to the cysteine residue at the dendrimer core. A structure-activity relationship study is reported concerning the role of negative charges in binding. Substituting glutamates (E) for glutamines (Q) in the outer branches of B1 to form N3 (AcQS)8(BQA)4(B-Amb-Y)(2)BCD-NH2 leads to stronger (K(a)=12.0 x 10(6) M(-1)) but slower (k(2)=67 M(-1) s(-1)) cobalamin binding. CD and FTIR spectra show that the dendrimers and their cobalamin complexes exist as random-coil structures without aggregation in solution. The hydrodynamic radii of the dendrimers determined by diffusion NMR either remains constant or slightly decreases upon binding to cobalamin; this indicates the formation of compact, presumably hydrophobically collapsed complexes.

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In vitro recombination mediated by G-quadruplexes.

Publication Date: 2010 Feb 15 PMID: 20014270
Authors: Boan, F. - Gomez-Marquez, J.
Journal: Chembiochem

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