ChemBioChem

Cover image for Vol. 8 Issue 6

April 16, 2007

Volume 8, Issue 6

Pages 581–690

  1. Cover Picture

    1. Top of page
    2. Cover Picture
    3. Graphical Abstract
    4. Communications
    5. Full Papers
    6. Previews
    1. Cover Picture: Synthesis of a 13C-Methyl-Group-Labeled Methionine Precursor as a Useful Tool for Simplifying Protein Structural Analysis by NMR Spectroscopy (ChemBioChem 6/2007) (page 581)

      Michael Fischer, Karin Kloiber, Johannes Häusler, Karin Ledolter, Robert Konrat and Walther Schmid

      Version of Record online: 29 MAR 2007 | DOI: 10.1002/cbic.200790013

      The cover picture shows the structure of the C-terminal SH2 domain of the signal transduction protein PLC-γ1. [4-13C]methylthio-2-oxobutanoates were used as precursors to introduce labeled methionines into the protein selectively. Synthesis of the labeled precursor was performed on a preparative scale by following a specially designed short and economical reaction sequence. Incorporation of the target protein into the SH2 domain was achieved by protein expression in E. coli BL21 (DE3) and confirmed by 13C,1H HSQC spectroscopy. The presence of selectively labeled methionine S-methyl groups in defined positions significantly facilitates structural and dynamic NMR analysis of the protein. Since this synthetic route could give access to a variety of labeling patterns within the carbon skeleton of methionine, the precursor is a powerful tool for answering structural biological questions. Further details on the synthesis, incorporation and NMR investigations can be found in the article by R. Konrat, W. Schmid, et al. on p. 610 ff. The cover picture was drawn by using MOLMOL.

  2. Graphical Abstract

    1. Top of page
    2. Cover Picture
    3. Graphical Abstract
    4. Communications
    5. Full Papers
    6. Previews
  3. Communications

    1. Top of page
    2. Cover Picture
    3. Graphical Abstract
    4. Communications
    5. Full Papers
    6. Previews
    1. Redox Potential of Azobenzene as an Amino Acid Residue in Peptides (pages 591–594)

      Cyril Boulègue, Markus Löweneck, Christian Renner and Luis Moroder

      Version of Record online: 16 MAR 2007 | DOI: 10.1002/cbic.200600495

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      Azobenzene is a common light switch. Azobenzene, as ω-amino acid derivative in peptides, exhibits redox properties that make it susceptible to reduction by thiols with concurrent thiol-induced enhanced thermal Z-to-E isomerization rates.

    2. A Simple and Efficient Photoaffinity Method for Proteomics of GTP-Binding Proteins (pages 595–598)

      Masaki Kaneda, Souta Masuda, Takenori Tomohiro and Yasumaru Hatanaka

      Version of Record online: 2 MAR 2007 | DOI: 10.1002/cbic.200600527

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      Multipurpose chemical. The diazirine-carrying GTP analogue shown in the scheme can be used for various purposes, such as, carbene-mediated cross-linking, tagging, and affinity isolation. Using this GTP analogue, we have developed a simple and efficient photoaffinity method that can be employed in proteomic analysis of GTP-binding proteins.

    3. Cloning and Heterologous Expression of the Aranciamycin Biosynthetic Gene Cluster Revealed a New Flexible Glycosyltransferase (pages 599–602)

      Andriy Luzhetskyy, Almuth Mayer, Jens Hoffmann, Stefan Pelzer, Meike Holzenkämper, Bettina Schmitt, Sven-Eric Wohlert, Andreas Vente and Andreas Bechthold

      Version of Record online: 14 MAR 2007 | DOI: 10.1002/cbic.200600529

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      Add sugar… Cloning and heterologous expression of the aranciamycin biosynthetic gene cluster revealed a new flexible glycosyltransferase, AraGT, which accepts different nucleotide-activated sugars (D-amicetose, L-rhodinose, L-rhamnose and L-axenose) The newly generated aranciamycin derivatives displayed antiproliferative activity against MaTu and MCF7 cells; this shows that the deoxysugar is important for anticancer activity.

    4. Optimization and Modifications of Aptamers Selected from Live Cancer Cell Lines (pages 603–606)

      Dihua Shangguan, Zhiwen Tang, Prabodika Mallikaratchy, Zeyu Xiao and Weihong Tan

      Version of Record online: 20 MAR 2007 | DOI: 10.1002/cbic.200600532

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      To cut a long story short. Single-stranded DNA aptamers that can recognize cancer cells specifically were optimized and modified. The modifications gave rise to aptamers that were much smaller in size (see scheme) and more stable in fetal bovine serum. This stability makes it possible to apply aptamers to in vivo applications, such as diagnosis and therapy.

    5. Converting Cytochrome c into a Peroxidase-Like Metalloenzyme by Molecular Design (pages 607–609)

      Zhong-Hua Wang, Ying-Wu Lin, Federico I. Rosell, Feng-Yun Ni, Hao-Jie Lu, Peng-Yuan Yang, Xiang-Shi Tan, Xiao-Yuan Li, Zhong-Xian Huang and A. Grant Mauk

      Version of Record online: 27 FEB 2007 | DOI: 10.1002/cbic.200600547

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      Even better than the real thing. Based on molecular design, an electron-transfer hemoprotein, cytochrome c, was converted into a peroxidase-like enzyme by introduction of a distal histidine into the heme pocket. The cytochrome c variants obtained, Tyr67His and Tyr67His/Met80Val (see figure), showed much higher peroxidase activities than wild-type cytochrome protein. More interestingly, the kcat/Km values of the new hemoproteins were higher than that of wild-type horseradish peroxidase.

    6. Synthesis of a 13C-Methyl-Group-Labeled Methionine Precursor as a Useful Tool for Simplifying Protein Structural Analysis by NMR Spectroscopy (pages 610–612)

      Michael Fischer, Karin Kloiber, Johannes Häusler, Karin Ledolter, Robert Konrat and Walther Schmid

      Version of Record online: 27 FEB 2007 | DOI: 10.1002/cbic.200600551

      Thumbnail image of graphical abstract

      Marked for life. For the characterization of larger proteins new NMR spectroscopy methods that focus on side-chain methyl groups have been developed by using selectively isotope-labeled precursor compounds. Here we report on the synthesis of a 13C-methyl-group-labeled methionine precursor on a preparative scale, and its incorporation into the SH2 domain of the protein PLC-γ1 (see scheme).

    7. FenF: Servicing the Mycosubtilin Synthetase Assembly Line in trans (pages 613–616)

      Zachary D. Aron, Pascal D. Fortin, Christopher T. Calderone and Christopher T. Walsh

      Version of Record online: 2 MAR 2007 | DOI: 10.1002/cbic.200600575

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      AT your service. We report the expression and characterization of FenF from mycosubtilin biosynthesis. This work represents the first kinetic and selectivity studies performed on an in trans AT domain servicing a polyketide synthase (PKS), and revealed a strong acyl-group specificity and broad promiscuity toward substrate carrier proteins. The lack of specificity in FenF-mediated malonyl transfer suggests that this protein might prove a powerful tool for combinatorial biosynthesis.

    8. Rational Manipulation of Carrier-Domain Geometry in Nonribosomal Peptide Synthetases (pages 617–621)

      Ye Liu and Steven D. Bruner

      Version of Record online: 5 MAR 2007 | DOI: 10.1002/cbic.200700010

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      Changing the assembly line. Nonribosomal peptide synthetases are multidomain enzyme assemblies responsible for the biosynthesis of a wide range of therapeutically important natural products. Here we describe an approach to construct stable, domain-specific conjugates of nonribosomal peptide synthetases by using synthetic analogues of coenzyme A.

    9. Nucleophile Selectivity of Chorismate-Utilizing Enzymes (pages 622–624)

      Olivier Kerbarh, Alessio Ciulli, Dimitri Y. Chirgadze, Tom L. Blundell and Chris Abell

      Version of Record online: 5 MAR 2007 | DOI: 10.1002/cbic.200700019

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      A comparison of the active sites from the crystal structures of the product complexes of salicylate synthase (Irp9) and the TrpE subunit of anthranilate synthase shows that they are identical, apart from residue Lys193 in Irp9, which is Gln262 in TrpE. 1H NMR spectroscopic analysis of the Irp9K193Q and TrpEQ262K mutants indicates that both residues have a key role in the initial nucleophilic attack at C2 of the common substrate chorismate.

  4. Full Papers

    1. Top of page
    2. Cover Picture
    3. Graphical Abstract
    4. Communications
    5. Full Papers
    6. Previews
    1. Structural Analysis of the Capsular Polysaccharide from Campylobacter jejuni RM1221 (pages 625–631)

      Michel Gilbert, Robert E. Mandrell, Craig T. Parker, Jianjun Li and Evgeny Vinogradov

      Version of Record online: 5 MAR 2007 | DOI: 10.1002/cbic.200600508

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      The complete structure of the CPS of C. jejuni RM1221 has been determined by using NMR spectroscopy, MS, and chemical methods. The CPS biosynthesis genes of C. jejuni RM1221 are conserved in other C. jejuni strains of the Penner serotype HS:53, including the serotype HS:53 reference strain RM3435. The xylulose (Xlu) substitution is nonstoichiometrical.

    2. ValC, a New Type of C7-Cyclitol Kinase Involved in the Biosynthesis of the Antifungal Agent Validamycin A (pages 632–641)

      Kazuyuki Minagawa, Yirong Zhang, Takuya Ito, Linquan Bai, Zixin Deng and Taifo Mahmud

      Version of Record online: 5 MAR 2007 | DOI: 10.1002/cbic.200600528

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      Two different paths to the same end-product. A new type of C7-cyclitol kinase that phosphorylates valienone and validone to afford their 7-phosphate derivatives has been identified in Streptomyces hygroscopicus var. jinggangensis 5008. The result provides new insights into the activity of this enzyme and its involvement in the biosynthesis of the antifungal agent validamycin A.

    3. Synthesis of a Novel Ceramide Analogue and its Use in a High-Throughput Fluorogenic Assay for Ceramidases (pages 642–648)

      Carmen Bedia, Josefina Casas, Virginie Garcia, Thierry Levade and Gemma Fabriàs

      Version of Record online: 16 MAR 2007 | DOI: 10.1002/cbic.200600533

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      Testing ceramidase activity. A reproducible and reliable procedure has been developed to measure the activity of ceramidases in microtiter plates, with both cell-free systems and in intact cells. The method is based on measuring the release of umbelliferone from a coumarinic substrate (1) after enzymatic hydrolysis, and further in situ chemical processing of the resulting aminodiol. The assay is of use in the screening of chemical libraries for the identification and characterization of acidic ceramidase inhibitors of therapeutical value.

    4. Using Selection to Identify and Chemical Microarray to Study the RNA Internal Loops Recognized by 6′-N-Acylated Kanamycin A (pages 649–656)

      Matthew D. Disney and Jessica L. Childs-Disney

      Version of Record online: 29 MAR 2007 | DOI: 10.1002/cbic.200600569

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      RNA chemical code enigma machine: A method has been developed to study the RNA secondary structure motifs that bind small molecules so as to facilitate the development of an RNA chemical code.

    5. Anomer-Selective Glycosidase Inhibition by 2-N-Alkylated 1-Azafagomines (pages 657–661)

      Oscar Lopez Lopez and Mikael Bols

      Version of Record online: 13 MAR 2007 | DOI: 10.1002/cbic.200700012

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      Raising the selectivity. 2-N-alkylated derivatives of azafagomine (2), made by reductive amination, were tested for inhibition of yeast α-glucosidase and of almond β-glucosidase. They were found to be stronger β- but weaker α-glucosidase inhibitors than either the parent compound or the related isofagomine (1), that is, far more anomer selective. The Ki(α-glu)/Ki(β-glu) values for the three inhibitors are given in brackets.

    6. A Water Molecule in the Stereospecificity Pocket of Candida Antarctica Lipase B Enhances Enantioselectivity towards Pentan-2-ol (pages 662–667)

      Valérie Léonard, Linda Fransson, Sylvain Lamare, Karl Hult and Marianne Graber

      Version of Record online: 27 FEB 2007 | DOI: 10.1002/cbic.200600479

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      Just add water. A water molecule bound in the stereospecificity pocket of lipase B from Candida antarctica causes enantioselective inhibition of pentan-2-ol acylation: the enantioselectivity is increased threefold. The bound water molecule loses all rotational freedom, equivalent to 37 J mol−1 K−1.

    7. Encapsulation of Hemoglobin in Mesoporous Silica (FSM)—Enhanced Thermal Stability and Resistance to Denaturants (pages 668–674)

      Yoko Urabe, Toru Shiomi, Tetsuji Itoh, Akiko Kawai, Tatsuo Tsunoda, Fujio Mizukami and Kengo Sakaguchi

      Version of Record online: 2 MAR 2007 | DOI: 10.1002/cbic.200600486

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      In a nutshell. The oligomeric protein hemoglobin (Hb) was successfully encapsulated in the pores of mesoporous silica (FSM: folded-sheet mesoporous material) that had a diameter of 7.5 nm (see scheme). The Hb–FSM conjugates showed increased thermal and chemical stability compared to native Hb. The mesopores seem to provide a favorable environment that prevents dissociation and denaturing of Hb, even under harsh conditions.

    8. Protonation Patterns in Tetracycline:Tet Repressor Recognition: Simulations and Experiments (pages 675–685)

      Alexey Aleksandrov, Juliane Proft, Winfried Hinrichs and Thomas Simonson

      Version of Record online: 16 MAR 2007 | DOI: 10.1002/cbic.200600535

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      A combination of free-energy simulations and crystallographic analysis has been used to investigate tetracycline (Tc) binding as a Tc:Mg2+ complex to the Tet repressor protein (TetR), and indicated that the ligand prefers an extended, zwitterionic state both in solution and in complexation with the protein. Tc is thus preorganized for binding, while the protein combines lock-and-key behavior for regions close to the ligand's amide, enolate, and ammonium groups with induced fit for regions close to the Mg2+ ion.

  5. Previews

    1. Top of page
    2. Cover Picture
    3. Graphical Abstract
    4. Communications
    5. Full Papers
    6. Previews
    1. You have free access to this content
      Preview: ChemBioChem 7/2007 (page 690)

      Version of Record online: 29 MAR 2007 | DOI: 10.1002/cbic.200790015

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