ChemBioChem

The Inner-Shell Film: An Immediate Structure Participating in Pearl Oyster Shell Formation.

Publication Date: 2008 May 15 PMID: 18481369
Authors: Yan, Z. - Ma, Z. - Zheng, G. - Feng, Q. - Wang, H. - Xie, L. - Zhang, R.
Journal: Chembiochem

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Preview: ChemBioChem 9/2008.

Publication Date: 2008 May 15 PMID: 18481368
Authors:
Journal: Chembiochem

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Spotlights on our sister journals: ChemBioChem 8/2008.

Publication Date: 2008 May 15 PMID: 18481367
Authors:
Journal: Chembiochem

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Rational Modification of Ligand-Binding Preference of Avidin by Circular Permutation and Mutagenesis.

Publication Date: 2008 May 15 PMID: 18481366
Authors: Maatta, J. A. - Airenne, T. T. - Nordlund, H. R. - Janis, J. - Paldanius, T. A. - Vainiotalo, P. - Johnson, M. S. - Kulomaa, M. S. - Hytonen, V. P.
Journal: Chembiochem

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Varied Active-Site Constraints in the Klenow Fragment of E. coli DNA Polymerase I and the Lesion-Bypass Dbh DNA Polymerase.

Publication Date: 2008 May 15 PMID: 18481365
Authors: Cramer, J. - Rangam, G. - Marx, A. - Restle, T.
Journal: Chembiochem

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A Caged Phosphopeptide-Based Approach for Photochemical Activation of Kinases in Living Cells.

Publication Date: 2008 May 15 PMID: 18481344
Authors: Kawakami, T. - Cheng, H. - Hashiro, S. - Nomura, Y. - Tsukiji, S. - Furuta, T. - Nagamune, T.
Journal: Chembiochem

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Rational Design of Highly Active and Selective Ligands for the alpha5beta1 Integrin Receptor.

Publication Date: 2008 May 15 PMID: 18481343
Authors: Heckmann, D. - Meyer, A. - Laufer, B. - Zahn, G. - Stragies, R. - Kessler, H.
Journal: Chembiochem

The inhibition of integrin function is a major challenge in medicinal chemistry. Potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (AMD). The subtype alpha5beta1 has recently been drawn into the focus of research because of its genuine role in angiogenesis. In our previous work we could demonstrate that the lack of structural information about the receptor could be overcome by a homology model based on the X-ray structure of the alphavbeta3 integrin subtype and the sequence similarities between both receptors. In this work, we describe the rational design and synthesis of high affinity alpha5beta1 binders, and the optimisation of selectivity against alphavbeta3 by means of extensive SAR studies and docking experiments. A first series of compounds based on the tyrosine scaffold resulted in affinities in the low and even subnanomolar range and selectivities of 400-fold against alphavbeta3. The insights about the structure-activity relationship gained from tyrosine-based ligands could be successfully transferred to ligands that bear an aza-glycine scaffold to yield alpha5beta1 ligands with affinities of ~1 nm and selectivities that exceed 10(4)-fold. The ligands have already been successfully employed as selective alpha5beta1 ligands in biological research and might serve as lead structures for antiangiogenic cancer therapy.

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Theoretical and Experimental Evaluation of a CYP106A2 Low Homology Model and Production of Mutants with Changed Activity and Selectivity of Hydroxylation.

Publication Date: 2008 May 15 PMID: 18481342
Authors: Lisurek, M. - Simgen, B. - Antes, I. - Bernhardt, R.
Journal: Chembiochem

Steroids are important pharmaceutically active compounds. In contrast to the liver drug-metabolising cytochrome P450s, which metabolise a variety of substrates, steroid hydroxylases generally display a rather narrow substrate specificity. It is therefore a challenging goal to change their regio- and stereoselectivity. CYP106A2 is one of only a few bacterial steroid hydroxylases and hydroxylates 3-oxo-Delta(4)-steroids mainly in 15beta-position. In order to gain insights into the structure and function of this enzyme, whose crystal structure is unknown, a homology model has been created. The substrate progesterone was then docked into the active site to predict which residues might affect substrate binding. The model was substantiated by using a combination of theoretical and experimental investigations. First, numerous computational structure evaluation tools assessed the plausibility of its protein geometry and its quality. Second, the model explains many key properties of common cytochrome P450s. Third, two sets of mutants have been heterologously expressed, and the influence of the mutations on the catalytic activity towards deoxycorticosterone and progesterone has been studied experimentally: the first set comprises six mutations located in the structurally variable regions of this enzyme that are very difficult to predict by cytochrome P450 modelling (K27R, I86T, E90V, I71T, D185G and I215T). For these positions, no participation in the active-site formation was predicted, or could be experimentally demonstrated. The second set comprises five mutants in substrate recognition site 6 (S394I, A395L, T396R, G397P and Q398S). For these residues, participation in active-site formation and an influence on substrate binding was predicted by docking. These mutants are based on an alignment with human CYP11B1, and in fact most of these mutants altered the active-site structure and the hydroxylation activity of CYP106A2 dramatically.

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The Fluorescent Amino Acid p-Cyanophenylalanine Provides an Intrinsic Probe of Amyloid Formation.

Publication Date: 2008 May 13 PMID: 18478525
Authors: Marek, P. - Gupta, R. - Raleigh, D. P.
Journal: Chembiochem

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Identification of Bacterial Carotenoid Cleavage Dioxygenase Homologues That Cleave the Interphenyl alpha,beta Double Bond of Stilbene Derivatives via a Monooxygenase Reaction.

Publication Date: 2008 May 13 PMID: 18478524
Authors: Marasco, E. K. - Schmidt-Dannert, C.
Journal: Chembiochem

Carotenoid cleavage oxygenases (CCOs), which are also referred to as carotenoid cleavage dioxygenases (CCDs) are a new class of nonheme iron-type enzymes that oxidatively cleave double bonds in the conjugated carbon chain of carotenoids. The oxidative cleavage mechanism of these enzymes is not clear, and both monooxygenase and dioxygenase mechanisms have been proposed for different carotenoid cleavage enzymes. CCOs have been described from plants, animals, fungi, and cyanobacteria, but little is known about their distribution and activities in bacteria other than cyanobacteria. We surveyed bacterial genome sequences for CCO homologues and report the characterization of CCO homologues that were identified in Novosphingobium aromaticivorans DSM 12444 (NOV1 and NOV2) and in Bradyrhizobium sp. (BRA-J and BRA-S). In vitro and in vivo assays with carotenoid and stilbene compounds were used to investigate the cleavage activities of the recombinant enzymes. The NOV enzymes cleaved the interphenyl alpha-beta double bond of stilbenes that had an oxygen functional group at the 4' carbon atom (e.g., resveratrol, piceatannol, and rhaponticin) to the corresponding aldehyde products. Carotenoids and apocarotenoids were not substrates for these enzymes. The two homologous enzymes from Bradyrhizobium sp. did not possess carotenoid or stilbene cleavage oxygenase activities, but showed activity with farnesol. To investigate whether the oxidative cleavage of stilbenes proceeds via a monooxygenase or dioxygenase reaction, oxygen-labeling studies were conducted with NOV2. Our labeling studies show that the double-bond cleavage of stilbenes occurs via a monooxygenase reaction mechanism.

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alpha-Lactosylceramide as a Novel "Sugar-Capped" CD1d Ligand for Natural Killer T Cells: Biased Cytokine Profile and Therapeutic Activities.

Publication Date: 2008 May 13 PMID: 18478523
Authors: Zhang, W. - Zheng, X. - Xia, C. - Perali, R. S. - Yao, Q. - Liu, Y. - Zheng, P. - Wang, P. G.
Journal: Chembiochem

The invariant natural killer T cells (iNKT) cells have emerged as an important regulator of immunity to infection, cancer, and autoimmune diseases. They can be activated by glycolipids that bind to CD1d. The most effective iNKT ligand reported to date is alpha-galactosylceramide (alpha-GalCer), which stimulates iNKT cells to secrete both Th-1 and Th-2 cytokines. Indiscriminate induction of both types of cytokines could limit the therapeutic potential of iNKT ligands, as Th-1 and Th-2 cytokines play different roles under physiological and pathological conditions. Therefore, a ligand with a biased cytokine-release profile would be highly desirable. Here, we report the synthesis and biological activity of alpha-lactosylceramide (alpha-LacCer). Our data demonstrate that alpha-LacCer can stimulate iNKT cells to proliferate and release cytokines, both in vitro and in vivo. Interestingly, while alpha-LacCer is approximately 1000-times less efficient than alpha-GalCer in inducing Th-1 cytokines, it is as potent as alpha-GalCer in the induction of Th-2 cytokines; therefore, alpha-LacCer is a novel compound that induces a biased cytokine release. Processing by beta-glycosidase was critical for alpha-LacCer activity. Moreover, in vivo experiments suggest that alpha-LacCer is at least as potent as alpha-GalCer in the treatment of tumors and experimental autoimmune encephalomyelitis.

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Dehydrophenylalanine (DeltaPhe) as a beta Breaker: Extended Structure Terminated by a DeltaPhe-Induced Turn in the Pentapeptide Boc-Phe1-Ala2-Ile3-DeltaPhe4-Ala5-OMe.

Publication Date: 2008 May 13 PMID: 18478512
Authors: Gupta, M. - Acharya, R. - Mishra, A. - Ramakumar, S. - Ahmed, F. - Chauhan, V. S.
Journal: Chembiochem

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In vivo Mutational Analysis of the Mupirocin Gene Cluster Reveals Labile Points in the Biosynthetic Pathway: the "Leaky Hosepipe" Mechanism.

Publication Date: 2008 May 8 PMID: 18465759
Authors: Wu, J. - Hothersall, J. - Mazzetti, C. - O'Connell, Y. - Shields, J. A. - Rahman, A. S. - Cox, R. J. - Crosby, J. - Simpson, T. J. - Thomas, C. M. - Willis, C. L.
Journal: Chembiochem

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.

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The Wall Teichoic Acid Polymerase TagF Efficiently Synthesizes Poly(glycerol phosphate) on the TagB Product Lipid III.

Publication Date: 2008 May 8 PMID: 18465758
Authors: Pereira, M. P. - Schertzer, J. W. - D'Elia, M. A. - Koteva, K. P. - Hughes, D. W. - Wright, G. D. - Brown, E. D.
Journal: Chembiochem

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Searching Combinatorial Libraries for Native Proteins with Novel Folds.

Publication Date: 2008 May 7 PMID: 18464233
Authors: Watkins, J. L. - Chaput, J. C.
Journal: Chembiochem

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Molecular Characterization of the NCoA-1-STAT 6 Interaction.

Publication Date: 2008 May 7 PMID: 18464232
Authors: Seitz, M. - Maillard, L. T. - Obrecht, D. - Robinson, J. A.
Journal: Chembiochem

Many protein-protein interactions involved in cell signalling, cell adhesion and regulation of transcription are mediated by short alpha-helical recognition motifs with the sequence Leu-Xaa-Xaa-Leu-Leu (LXXLL, where Xaa is any amino acid). Originally observed in cofactors that interact with hormone-activated nuclear receptors, LXXLL motifs are now known to occur in many transcription factors, including the STAT family, which transmit signals from activated cytokine receptors at the cell surface to target genes in the nucleus. STAT 6 becomes activated in response to IL-4 and IL-13, which regulate immune and anti-inflammatory responses. Structural studies have revealed how an LXXLL motif located in 2.5 turns of an alpha-helical peptide derived from STAT 6 provide contacts through the leucine side chains to the coactivator of transcription, NCoA-1. However, since many protein-protein interactions are mediated by LXXLL motifs, it is important to understand how specificity is achieved in this and other signalling pathways. Here, we show that energetically important contacts between STAT 6 and NCoA-1 are made in residues that flank the LXXLL motif, including the underlined residues in the sequence LLPPTEQDLTKLL. We also demonstrate how the affinity for NCoA-1 of peptides derived from this region of STAT 6 can be significantly improved by optimising knobs-into-holes contacts on the surface of the protein. The results provide important new insights into the origins of binding specificity, and might be of practical value in the design of novel small-molecule inhibitors of this important protein-protein interaction.

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NMR-Guided Fragment-Based Approach for the Design of AAC(6')-Ib Ligands.

Publication Date: 2008 May 7 PMID: 18464231
Authors: Lombes, T. - Begis, G. - Maurice, F. - Turcaud, S. - Lecourt, T. - Dardel, F. - Micouin, L.
Journal: Chembiochem

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Covalent Fluorescence Labeling of His-Tagged Proteins on the Surface of Living Cells.

Publication Date: 2008 May 6 PMID: 18461582
Authors: Hintersteiner, M. - Weidemann, T. - Kimmerlin, T. - Filiz, N. - Buehler, C. - Auer, M.
Journal: Chembiochem

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Synthesis and Transfection Activity of New Cationic Phosphoramidate Lipids: High Efficiency of an Imidazolium Derivative.

Publication Date: 2008 May 2 PMID: 18454443
Authors: Mevel, M. - Breuzard, G. - Yaouanc, J. J. - Clement, J. C. - Lehn, P. - Pichon, C. - Jaffres, P. A. - Midoux, P.
Journal: Chembiochem

In an effort to enhance the gene-transfer efficiencies of cationic lipids and to decrease their toxicities, a series of new phosphoramidate lipids with chemical similarity to cell membrane phospholipids was synthesised. These lipids contained various cationic headgroups, such as arginine methyl ester, lysine methyl ester, homoarginine methyl ester, ethylenediamine, diaminopropane, guanidinium and imidazolium. Their transfection abilities, either alone or with the co-lipid DOPE, were evaluated in HEK293-T7 cells. We found that imidazolium lipophosphoramidate 7 a/DOPE lipoplexes gave the most efficient transfection with low toxicity (15 %). The luciferase activity was 100 times higher than that obtained with DOTAP/DOPE lipoplexes. The size, zeta potential, pDNA-liposome interactions and cellular uptakes of the lipoplexes were determined. No definitive correlation between the zeta potential values and the transfection efficiencies could be established, but the uptake of lipoplexes by the cells was correlated with their final transfection efficiencies. Our results show that imidazolium phosphoramidate lipids constitute a potential new class of cationic lipids for gene transfer.

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Arginine Dynamics in a Membrane-Bound Cationic Beta-Hairpin Peptide from Solid-State NMR.

Publication Date: 2008 Apr 29 PMID: 18442147
Authors: Tang, M. - Waring, A. J. - Hong, M.
Journal: Chembiochem

The site-specific motion of Arg residues in a membrane-bound disulfide-linked antimicrobial peptide, protegrin-1 (PG-1), was investigated by using magic-angle-spinning solid-state NMR spectroscopy to better understand the membrane insertion and lipid interaction of this cationic membrane-disruptive peptide. The C-H and N-H dipolar couplings and (13)C chemical shift anisotropies were measured in the anionic POPE/POPG membrane, and were found to be reduced from the rigid-limit values by varying extents; this indicates the presence of segmental motion. An Arg residue at the beta-turn region of the peptide showed much weaker spin interactions, which indicates larger amplitudes of motion than an Arg residue in the beta-strand region of the peptide. This is consistent with the exposure of the beta turn to the membrane surface and the immersion of the beta strand in the hydrophobic middle of the membrane, and supports the previously proposed oligomerization of the peptide into beta barrels in the anionic membrane. The (13)C T(2) and (1)H T(1rho) relaxation times indicate that the beta-turn backbone undergoes large-amplitude intermediate-timescale motion in the fluid phase of the membrane; this causes significant line broadening and loss of spectral intensity. This study illustrates the strong correlation between the dynamics and structure of membrane proteins, and the capability of solid-state NMR spectroscopy to provide detailed information on site-specific dynamics in complex membrane-protein assemblies.

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Contribution of Fluorophores to Protein Kinase C FRET Probe Performance.

Publication Date: 2008 Apr 29 PMID: 18442146
Authors: Jost, C. A. - Reither, G. - Hoffmann, C. - Schultz, C.
Journal: Chembiochem

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Spotlights on our sister journals: ChemBioChem 7/2008.

Publication Date: 2008 May 5 PMID: 18438956
Authors:
Journal: Chembiochem

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Preview: ChemBioChem 8/2008.

Publication Date: 2008 May 5 PMID: 18438955
Authors:
Journal: Chembiochem

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Generation of shRNA Pool Library: A Revision of the Biological Technique from the Viewpoint of Chemistry.

Publication Date: 2008 Apr 24 PMID: 18435449
Authors: Zhou, D. - Wang, C. - Zhang, J. - Bliesath, J. - He, Q. S. - Ke, N. - Yu, D. - Li, Q. - Zhang, L. H. - Wong-Staal, F.
Journal: Chembiochem

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Cellular Dynamics of Ku: Characterization and Purification of Ku-eGFP.

Publication Date: 2008 Apr 24 PMID: 18435448
Authors: Merkle, D. - Zheng, D. - Ohrt, T. - Crell, K. - Schwille, P.
Journal: Chembiochem

Ku is a predominantly nuclear protein that functions as a DNA double-strand-break (DSB) binding protein and regulatory subunit of the DNA-dependent protein kinase (DNA-PK). DNA-PK is involved in synapsis and remodeling of broken DNA ends during nonhomologous end-joining (NHEJ) of DNA DSBs. It has also recently been demonstrated that Ku plays roles in cytoplasmic and membrane processes, namely: interaction with matrix metalloproteinase 9, acting as a co-receptor for parvoviral infection, and also interacting with cell polarity protein, Par3. We present a method for creating stable expression of Ku-eGFP in CHO cells and extend the procedure to purify Ku-eGFP for in vitro assaying. We demonstrated that Ku-eGFP localizes to the nucleus of HeLa cells upon microinjection into the cytoplasm as well as localizing to laser induced DNA damage. We also characterized the diffusional dynamics of Ku in the nucleus and in the cytoplasm using fluorescence correlation spectroscopy (FCS). The FCS data suggest that whereas the majority of Ku (70 %) in the nucleus is mobile and freely diffusing, in a cellular context, there also exists a significant slow process fraction (30 %). Strikingly, in the cytoplasm, this immobile/slow moving fraction is even more pronounced (45 %).

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Synthesis and Screening of an Oroidin Library against Pseudomonas aeruginosa Biofilms.

Publication Date: 2008 Apr 22 PMID: 18431726
Authors: Richards, J. J. - Ballard, T. E. - Huigens, R. W. 3rd - Melander, C.
Journal: Chembiochem

A 50-compound library based on the marine natural product oroidin was synthesized and assayed for anti-biofilm activity against PAO1 and PA14, two strains of the medically relevant gamma-proteobacterium Pseudomonas aeruginosa. Through structure-activity relationship (SAR) analysis of analogues based on the oroidin template, several conclusions can be drawn as to what structural properties of the synthetic derivatives are necessary to elicit a biological response. Notably, the most active analogues identified were those that contained a 2-aminoimidazole (2-AI) motif and a dibrominated pyrrolecarboxamide subunit. Here we disclose the synthesis and subsequently determined biological activity of this unique class of compounds as inhibitors of biofilm formation that have no direct antibiotic effect.

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Tyl1a, a TDP-6-deoxy-D-xylo-4-hexulose 3,4-isomerase from Streptomyces fradiae: Structure Prediction, Mutagenesis and Solvent Isotope Incorporation Experiments to Investigate Reaction Mechanism.

Publication Date: 2008 Apr 21 PMID: 18425854
Authors: Tello, M. - Rejzek, M. - Wilkinson, B. - Lawson, D. M. - Field, R. A.
Journal: Chembiochem

Understanding the structure and mechanism of sugar nucleotide processing enzymes is invaluable in the generation of designer enzymes for biotransformation, for instance, in connection with engineering antibiotic glycosylation. In this study, homology modelling and mechanistic comparison to the structurally related RmlC epimerase family has been used to identify and assign functions to active-site residues in the Tyl1a-catalysed keto-sugar nucleotide isomerisation process. Tyl1a His63 is implicated as the base that initiates the isomerisation process by substrate C-3 deprotonation, with Arg109 stabilising the resulting enolate. Subsequent O-3 deprotonation (potentially by His65) and C-4 protonation (potentially by Tyr49) complete the isomerisation process.

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Synthesis and application of fluorescein- and biotin-labeled molecular probes for the chemokine receptor CXCR4.

Publication Date: 2008 May 5 PMID: 18412193
Authors: Oishi, S. - Masuda, R. - Evans, B. - Ueda, S. - Goto, Y. - Ohno, H. - Hirasawa, A. - Tsujimoto, G. - Wang, Z. - Peiper, S. C. - Naito, T. - Kodama, E. - Matsuoka, M. - Fujii, N.
Journal: Chembiochem

The design, synthesis, and bioevaluation of fluorescence- and biotin-labeled CXCR4 antagonists are described. The modification of D-Lys8 at an epsilon-amino group in the peptide antagonist Ac-TZ14011 derived from polyphemusin II had no significant influence on the potent binding of the peptide to the CXCR4 receptor. The application of the labeled peptides in flow cytometry and confocal microscopy studies demonstrated the selectivity of their binding to the CXCR4 receptor, but not to CXCR7, which was recently reported to be another receptor for stromal cell-derived factor 1 (SDF-1)/CXCL12.

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Analysis of the tetronomycin gene cluster: insights into the biosynthesis of a polyether tetronate antibiotic.

Publication Date: 2008 May 5 PMID: 18404760
Authors: Demydchuk, Y. - Sun, Y. - Hong, H. - Staunton, J. - Spencer, J. B. - Leadlay, P. F.
Journal: Chembiochem

The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open-reading frames. Based on sequence comparison to related biosynthetic gene clusters, the majority of these can be assigned a plausible role in TMN biosynthesis. The identity of the cluster, and the requirement for a number of individual genes, especially those hypothesised to contribute a glycerate unit to the formation of the tetronate ring, were confirmed by specific gene disruption. However, two large genes that are predicted to encode together a multifunctional PKS of a highly unusual type seem not to be involved in this pathway since deletion of one of them did not alter tetronomycin production. Unlike previously characterised polyether PKS systems, oxidative cyclisation appears to take place on the modular PKS rather than after transfer to a separate carrier protein, while tetronate ring formation and concomitant chain release share common mechanistic features with spirotetronate biosynthesis.

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A simple method for preparing peptide C-terminal thioacids and their application in sequential chemoenzymatic ligation.

Publication Date: 2008 May 5 PMID: 18398882
Authors: Tan, X. H. - Zhang, X. - Yang, R. - Liu, C. F.
Journal: Chembiochem

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Globotriose-functionalized gold nanoparticles as multivalent probes for Shiga-like toxin.

Publication Date: 2008 May 5 PMID: 18398881
Authors: Chien, Y. Y. - Jan, M. D. - Adak, A. K. - Tzeng, H. C. - Lin, Y. P. - Chen, Y. J. - Wang, K. T. - Chen, C. T. - Chen, C. C. - Lin, C. C.
Journal: Chembiochem

Compared to monovalent carbohydrates, multivalent carbohydrate ligands exhibit significantly enhanced binding affinities to their interacting proteins. Here, we report globotriose (P(k) ligand)-functionalized gold nanoparticle (AuNP) probes for the investigation of multivalent interactions with the B(5) subunit of Shiga-like toxin I (B-Slt). Six P(k)-ligand-encapsulated AuNPs (P(k)-AuNPs) of varying particle size and linker length were synthesized and evaluated for their potential as multivalent affinity probes by using a surface plasmon resonance competition assay. The affinity of these probes for the interacting proteins was greatly affected by nanoparticle size, linker length, and ligand density on nanoparticle surface. For example, the 20-nm 20-P(k)-l-AuNP, which had a relatively long linker showed a >10(8)-fold increase in affinity compared with the mono P(k) ligand. This intrinsic high-affinity AuNP probe specifically captured the recombinant B-Slt from Escherichia coli lysate, and the resulting purity of the B-Slt was >95 %. We also developed a robust P(k)-AuNP-based detection method for Slt-I by combining the technique with silver enhancement.

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Protein surface recognition: structural characterisation of cytochrome c-porphyrin complexes.

Publication Date: 2008 May 5 PMID: 18389511
Authors: Crowley, P. B. - Ganji, P. - Ibrahim, H.
Journal: Chembiochem

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Fluorescent agonists for the torpedo nicotinic acetylcholine receptor.

Publication Date: 2008 May 5 PMID: 18386276
Authors: Krieger, F. - Mourot, A. - Araoz, R. - Kotzyba-Hibert, F. - Molgo, J. - Bamberg, E. - Goeldner, M.
Journal: Chembiochem

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.

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Probing the hydrophobic pocket of the active site in the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by variable stereoselective alkane hydroxylation and olefin epoxidation.

Publication Date: 2008 May 5 PMID: 18383583
Authors: Ng, K. Y. - Tu, L. C. - Wang, Y. S. - Chan, S. I. - Yu, S. S.
Journal: Chembiochem

pMMO from M. capsulatus (Bath) oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides. According to experiments performed with cryptically chiral ethane and D,L-[2-(2)H(1),3-(2)H(1)]butane, the reactions proceed through the concerted O-atom insertion mechanism. However, when propene and but-1-ene are used as epoxidation substrates, the enantiomeric excesses (ees) of the enzymatic products are only 18 and 37 %, respectively. This relatively poor stereoselectivity in the enzymatic epoxidation presumably reflects low stereochemical differentiation between the re and si faces in the hydrophobic pocket of the active site. Further insights into the reaction mechanism are now provided by studies on trans-but-2-ene, which reveal only the D,L-2,3-dimethyloxirane products, and on cis-but-2-ene, which yield only the meso product. These observations indicate that the enzymatic epoxidation indeed proceeds through electrophilic syn addition. To achieve better facial selectivity, we have also used 3,3,3-trifluoroprop-1-ene as the substrate. The products obtained are 90 % (2S)-oxirane. When 1,1,1-trifluoropropane is the substrate, the hydroxylation at the 2-carbon exhibits an inverse chiral selectivity relative to that seen with normal butane, if we consider the size of the CF(3) group in the fluorinated propane to be comparable to one of the ethyl groups in butane. These experiments are beginning to delineate the factors that influence the orientations of various substrates in the hydrophobic cavity of the active site in the enzyme.

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The inner-shell film: an immediate structure participating in pearl oyster shell formation.

Publication Date: 2008 May 5 PMID: 18383500
Authors: Yan, Z. - Ma, Z. - Zheng, G. - Feng, Q. - Wang, H. - Xie, L. - Zhang, R.
Journal: Chembiochem

In mollusks, the inner shell film is located in the shell-mantle zone and it is important in shell formation. In this study, we found that the film was composed of two individual films under certain states and some columnar structures were observed between the two individual films. The inner shell film was separated with the process of ethylenediaminetetraacetic acid (EDTA) treatment and the film proteins were extracted. Amino acid analysis showed that the film proteins may consist of shell framework proteins. The calcite crystallization experiment showed that the film proteins could inhibit the growth of calcite, while the CaCO(3) precipitation experiment showed that the film proteins could accelerate the rate of CaCO(3) precipitation. All these results suggested that the film plays an important role in shell formation. It may facilitate the aragonite formation by inhibiting the growth of calcite and accelerate the shell growth by promoting the precipitation of CaCO(3) crystals.

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A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A.

Publication Date: 2008 May 5 PMID: 18383241
Authors: Boersma, Y. L. - Droge, M. J. - van der Sloot, A. M. - Pijning, T. - Cool, R. H. - Dijkstra, B. W. - Quax, W. J.
Journal: Chembiochem

In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects beta7 and alphaE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.

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Reinvestigation of a cyclic dipeptide N-prenyltransferase reveals rearrangement of N-1 prenylated indole derivatives.

Publication Date: 2008 May 5 PMID: 18383240
Authors: Ruan, H. L. - Yin, W. B. - Wu, J. Z. - Li, S. M.
Journal: Chembiochem

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An amphiphilic bisporphyrin and its Yb(III) complex: development of a bifunctional photodynamic therapeutic and near-infrared tumor-imaging agent.

Publication Date: 2008 May 5 PMID: 18383057
Authors: Jiang, F. L. - Poon, C. T. - Wong, W. K. - Koon, H. K. - Mak, N. K. - Choi, C. Y. - Kwong, D. W. - Liu, Y.
Journal: Chembiochem

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Rational modification of ligand-binding preference of avidin by circular permutation and mutagenesis.

Publication Date: 2008 May 5 PMID: 18381715
Authors: Maatta, J. A. - Airenne, T. T. - Nordlund, H. R. - Janis, J. - Paldanius, T. A. - Vainiotalo, P. - Johnson, M. S. - Kulomaa, M. S. - Hytonen, V. P.
Journal: Chembiochem

Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4-->3) that lacks the loop between beta-strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4-->3 even further, we mutated asparagine 118 on the bottom of the ligand-binding pocket to methionine, which simultaneously caused a significant drop in biotin-binding affinity. The X-ray structure of cpAvd4--> 3(N118M) allows an understanding of the effect of mutation to biotin-binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion-fold between wild-type avidin and cpAvd4-->3(N118M). To demonstrate the versatility of the cpAvd4-->3 construct, we have shown that it is possible to link cpAvd4-->3 and cpAvd5-->4 to form the dual-chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self-organising nanoscale avidin building blocks.

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Highly active ansamitocin derivatives: mutasynthesis using an AHBA-blocked mutant.

Publication Date: 2008 May 5 PMID: 18381586
Authors: Taft, F. - Brunjes, M. - Floss, H. G. - Czempinski, N. - Grond, S. - Sasse, F. - Kirschning, A.
Journal: Chembiochem

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Design of lectin mimetics.

Publication Date: 2008 May 5 PMID: 18366054
Authors: Mazik, M.
Journal: Chembiochem

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Catalytic promiscuity of halohydrin dehalogenase and its application in enantioselective epoxide ring opening.

Publication Date: 2008 May 5 PMID: 18357593
Authors: Hasnaoui-Dijoux, G. - Majeric Elenkov, M. - Lutje Spelberg, J. H. - Hauer, B. - Janssen, D. B.
Journal: Chembiochem

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Naturally occurring small-molecule inhibitors of hedgehog/GLI-mediated transcription.

Publication Date: 2008 May 5 PMID: 18357592
Authors: Hosoya, T. - Arai, M. A. - Koyano, T. - Kowithayakorn, T. - Ishibashi, M.
Journal: Chembiochem

The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. To search for Hh/GLI inhibitors, we screened for naturally occurring inhibitors of the transcriptional activator GLI1 by using a cell-based assay. We identified zerumbone (1), zerumbone epoxide (2), staurosporinone (9), 6-hydroxystaurosporinone (10), arcyriaflavin C (11) and 5,6-dihydroxyarcyriaflavin A (12) as inhibitors of GLI-mediated transcription. In addition, we isolated physalins F (17) and B (18) from Physalis minima, which are also potent inhibitors. These compounds also inhibited GLI2-mediated transactivation. Semiquantitative RT-PCR and Western blotting analysis further revealed that 1, 9, 17, and 18 decreased Hh-related component expressions. We also show that inhibitors of GLI-mediated transactivation reduce the level of the antiapoptosis Bcl2 expression. Finally, these identified compounds were cytotoxic to PANC1 pancreatic cancer cells, which express Hh/GLI components. These results strongly suggest that the cytotoxicity of the compounds to PANC1 cells correlates with their inhibition of GLI-mediated transcription.

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Structure and mechanistic implications of a tryptophan synthase quinonoid intermediate.

Publication Date: 2008 May 5 PMID: 18351684
Authors: Barends, T. R. - Domratcheva, T. - Kulik, V. - Blumenstein, L. - Niks, D. - Dunn, M. F. - Schlichting, I.
Journal: Chembiochem

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Investigation of DNA-protein cross-link formation between lysozyme and oxanine by mass spectrometry.

Publication Date: 2008 May 5 PMID: 18351683
Authors: Chen, H. J. - Chiu, W. L. - Lin, W. P. - Yang, S. S.
Journal: Chembiochem

Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion product originating from the guanine base through exposure to nitric oxide, nitrous acid, or N-nitrosoindoles. Oxanine was found to mediate formation of DNA-protein cross-links (DPCs) in the cell extract. We have previously characterized two DNA-protein cross-links from the reaction between Oxa and glutathione: namely, the thioester and the amide. In this study, lysozyme was used to study site-specific modification on protein by Oxa moieties in DNA. With the aid of nanoLC coupled with nanospray ionization tandem mass spectrometry, addition of Oxa was found at Lys13, Lys97, Lys116, Ser85, and Ser86 of lysozyme when it was treated with 2'-deoxyoxanosine (dOxo). Furthermore, incubation of lysozyme with Oxa-containing calf thymus DNA, produced by treating DNA with nitrous acid, led to lysozyme modification at Lys116, Ser85, and Ser86. Interestingly, none of the cysteine residues was modified by dOxo, in contrast with our previous findings that dOxo reacted with oxidized glutathione disulfide, forming the thioester. This might be due to the half-life of the dOxo-derived thioester being 2.2 days at the pH of incubation. Furthermore, the sites of modifications on lysozyme are in good agreement with the solvent accessibility of the residues. Since repair of Oxa-derived DPCs has not been extensively investigated, these results suggest that these stable DPCs might represent important forms of cellular damage caused by reactive nitrogen species involved in inflammationrelated diseases.

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Novel DNA catalysts based on G-quadruplex recognition.

Publication Date: 2008 May 5 PMID: 18350529
Authors: Tang, Z. - Goncalves, D. P. - Wieland, M. - Marx, A. - Hartig, J. S.
Journal: Chembiochem

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An ESI-MS/MS method for screening of small-molecule mixtures against glycogen synthase kinase-3beta (GSK-3beta).

Publication Date: 2008 May 5 PMID: 18348127
Authors: Partserniak, I. - Werstuck, G. - Capretta, A. - Brennan, J. D.
Journal: Chembiochem

Glycogen synthase kinase-3beta (GSK-3beta) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of gamma-(32)P-radiolabelled isotopes into substrate peptides. The requirement to detect hyperphosphorylation of a primed substrate poses a particular challenge for development of a high-throughput screening assay, as many current kinase assays are designed to produce a signal in the presence of any phosphorylation site, and thus are only suitable for beta-unphosphorylated substrates. Herein, we have developed an electrospray-ionization tandem mass spectrometry (ESI-MS/MS) assay to allow for direct detection of a hyperphosphorylated product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3beta. Optimum reaction conditions (level of Mg(2+), buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3beta and allow for substrate and product quantification through ESI/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3beta activity from reaction volumes of approximately 40 microL and that it can be used to assess IC(50) values and the site of action of known inhibitors. It also can be used for automated screening of small-molecules mixtures to identify inhibitors of GSK-3beta.

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Synthetic strategy of nonreducing iterative polyketide synthases and the origin of the classical "starter-unit effect".

Publication Date: 2008 May 5 PMID: 18338425
Authors: Crawford, J. M. - Vagstad, A. L. - Whitworth, K. P. - Ehrlich, K. C. - Townsend, C. A.
Journal: Chembiochem

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A sensitive and selective near-infrared fluorescent probe for mercuric ions and its biological imaging applications.

Publication Date: 2008 May 5 PMID: 18338355
Authors: Tang, B. - Cui, L. J. - Xu, K. H. - Tong, L. L. - Yang, G. W. - An, L. G.
Journal: Chembiochem

A new mercury(II) near-infrared region fluorescent probe 3,9-dithia-6-monoazaundecane-tricarbocyanine has been designed and synthesized. It consists of two functional moieties: the tricarbocyanine performs as the near-infrared region fluorophore, and the 3,9-dithia-6-monoazaundecane acts as the selected binding site for metal ions. The near-IR excitation and emission profiles of the probe can minimize cell and tissue damage and avoid native fluorescence from natural cellular species. It exhibits fluorescence increase upon the binding of the Hg(2+) based on the inhibition of the photoinduced electron transfer quenching mechanism. Excellent sensitivity and selectivity for mercuric ions are observed with this probe. The value of the system is demonstrated by its use in monitoring the real-time uptake of Hg(2+) within HepG2 cells and five day old zebrafish. The synthesis and remarkable properties of it help to extend the development of metal ions fluorescent probes for biological applications.

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Riboswitches for enhancing target gene expression in eukaryotes.

Publication Date: 2008 May 5 PMID: 18338354
Authors: Yamauchi, T. - Miyoshi, D. - Kubodera, T. - Ban, M. - Nishimura, A. - Sugimoto, N.
Journal: Chembiochem

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