• Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

      el-Enshasy, Hesham; Kleine, Joachim; Rinas, Ursula; Helmholtz zENTRUM FÜR iNFEKTIONSFORSCHUNG (2006-10)
    • Biomacromolecular interactions, assemblies and machines: a structural view.

      Heinz, Dirk W; Weiss, Manfred S; Wendt, K Ulrich (Wiley and Sons, 2006-01-01)
    • Biosynthesis of the repeating units of the exopolysaccharides amylovoran from Erwinia amylovora and stewartan from Pantoea stewartii

      Langlotz, Christine; Schollmeyer, Martin; Coplin, David L.; Nimtz, Manfred; Geider, Klaus; Max-Planck-Institute for Cell Biology, Ladenburg, Germany (2012-06-27)
    • Callyaerin G, a new cytotoxic cyclic peptide from the marine sponge Callyspongia aerizusa

      Ibrahim, Sabrin R. M.; Edrada-Ebel, RuAngelie; Mohamed,Gamal A.; Youssef, Diaa T. A; Wray, Victor; Proksch, Peter; Helmholz Centre for Infection research, D-38124 Braunschweig, Germany (Arkat, 2008-04-16)
    • Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

      Schulze, Jörg O; Masoumi, Ava; Nickel, Daniel; Jahn, Martina; Jahn, Dieter; Schubert, Wolf-Dieter; Heinz, Dirk W; Division of Structural Biology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany. (2006-09-01)
      Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.
    • Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx.

      Wilke, Sonja; Krausze, Joern; Büssow, Konrad; Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstr, 7, 38124 Braunschweig, Germany. konrad@buessow.com. (2012)
      ABSTRACT:
    • Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa.

      Hagelueken, Gregor; Wiehlmann, Lutz; Adams, Thorsten M; Kolmar, Harald; Heinz, Dirk W; Tümmler, Burkhard; Schubert, Wolf-Dieter; Molecular Host-Pathogen Interactions, Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2007-07-24)
      Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon metabolism to the membrane-bound alkane hydroxylases AlkB1 and AlkB2. The RdxR-Rdx system also is crucial as part of the oxidative stress response in archaea or anaerobic bacteria. The redox couple has been analyzed in detail as a model system for ET processes. We have solved the structure of RdxR of P. aeruginosa both alone and in complex with Rdx, without the need for cross-linking, and both structures were refined at 2.40- and 2.45-A resolution, respectively. RdxR consists of two cofactor-binding domains and a C-terminal domain essential for the specific recognition of Rdx. Only a small number of direct interactions govern mutual recognition of RdxR and Rdx, corroborating the transient nature of the complex. The shortest distance between the redox centers is observed to be 6.2 A.
    • Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases.

      Storbeck, Sonja; Saha, Sayantan; Krausze, Joern; Klink, Björn U; Heinz, Dirk W; Layer, Gunhild; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany. (2011-07-29)
      During the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-L-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-L-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a "puckered" conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.
    • Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite.

      Singh, Bishal K; Sattler, Julia M; Chatterjee, Moon; Huttu, Jani; Schüler, Herwig; Kursula, Inari (2011-08-12)
      Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.
    • Determinants of ligand binding and catalytic activity in the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase.

      Raasakka, Arne; Myllykoski, Matti; Laulumaa, Saara; Lehtimäki, Mari; Härtlein, Michael; Moulin, Martine; Kursula, Inari; Kursula, Petri; CSSB, Centre for Structural Systems Biology, Notekestr. 85, 22607 Hamburg, Germany. (2015)
      2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enzyme highly abundant in the central nervous system myelin of terrestrial vertebrates. The catalytic domain of CNPase belongs to the 2H phosphoesterase superfamily and catalyzes the hydrolysis of nucleoside 2',3'-cyclic monophosphates to nucleoside 2'-monophosphates. The detailed reaction mechanism and the essential catalytic amino acids involved have been described earlier, but the roles of many amino acids in the vicinity of the active site have remained unknown. Here, several CNPase catalytic domain mutants were studied using enzyme kinetics assays, thermal stability experiments, and X-ray crystallography. Additionally, the crystal structure of a perdeuterated CNPase catalytic domain was refined at atomic resolution to obtain a detailed view of the active site and the catalytic mechanism. The results specify determinants of ligand binding and novel essential residues required for CNPase catalysis. For example, the aromatic side chains of Phe235 and Tyr168 are crucial for substrate binding, and Arg307 may affect active site electrostatics and regulate loop dynamics. The β5-α7 loop, unique for CNPase in the 2H phosphoesterase family, appears to have various functions in the CNPase reaction mechanism, from coordinating the nucleophilic water molecule to providing a binding pocket for the product and being involved in product release.
    • Discontinuous and continuous separation of the monomeric and dimeric forms of human bone morphogenetic protein-2 from renaturation batches.

      Gueorguieva, Ludmila; Vallejo, Luis Felipe; Rinas, Ursula; Seidel-Morgenstern, Andreas; Otto-von-Guericke-Universität Magdeburg, Institut für Verfahrenstechnik, PO Box 4120, D-39106 Magdeburg, Germany. (2006-12-01)
      Bone morphogenetic protein-2 (BMP-2) is one of the most interesting of the approximately 14 BMPs which belong to the transforming-growth-factor-beta (TGF-beta) superfamily. BMP-2 induces bone formation and thus plays an important role as a pharmaceutical protein. Recently, rhBMP-2 has been produced in form of inactive inclusion bodies in Escherichia coli. After solubilization and renaturation the biologically active dimeric form of rhBMP-2 can be generated. However, inactive monomers of BMP-2 are also formed during the renaturation process which must be separated from the active dimeric BMP-2. The purpose of this paper is to present: (a) results of an experimental study of a chromatographic separation of the monomeric and dimeric forms; and (b) a concept for a continuous counter-current simulated moving bed (SMB) process. The capacity of heparin as stationary phase was estimated for different salt concentrations in the mobile phase. A simulation study of a three-zone SMB process was performed applying a two step salt gradient. The results reveal the potential of the process for the purification of the dimeric BMP-2.
    • Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling.

      Wodke, Judith A H; Puchałka, Jacek; Lluch-Senar, Maria; Marcos, Josep; Yus, Eva; Godinho, Miguel; Gutiérrez-Gallego, Ricardo; dos Santos, Vitor A P Martins; Serrano, Luis; Klipp, Edda; et al. (2013)
      Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.
    • Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis.

      Schulze, Jörg O; Schubert, Wolf-Dieter; Moser, Jürgen; Jahn, Dieter; Heinz, Dirk W (2006-05-19)
      Glutamate-1-semialdehyde 2,1-aminomutase (GSAM) is the second enzyme in the C(5) pathway of tetrapyrrole biosynthesis found in most bacteria, in archaea and in plants. It catalyzes the transamination of glutamate-1-semialdehyde to 5-aminolevulinic acid (ALA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. We present the crystal structure of GSAM from the thermophilic cyanobacterium Thermosynechococcus elongatus (GSAM(Tel)) in its PLP-bound form at 2.85A resolution. GSAM(Tel) is a symmetric homodimer, whereas GSAM from Synechococcus (GSAM(Syn)) has been described as asymmetric. The symmetry of GSAM(Tel) thus challenges the previously proposed negative cooperativity between monomers of this enzyme. Furthermore, GSAM(Tel) reveals an extensive flexible region at the interface of the proposed complex of GSAM with glutamyl-tRNA reductase (GluTR), the preceding enzyme in tetrapyrrole biosynthesis. Compared to GSAM(Syn), the monomers of GSAM(Tel) are rotated away from each other along the dimerization interface by 10 degrees . The associated flexibility of GSAM may be essential for complex formation with GluTR to occur. Unexpectedly, we find that GSAM is structurally related to 5-aminolevulinate synthase (ALAS), the ALA-producing enzyme in the Shemin pathway of alpha-proteobacteria and non-plant eukaryotes. This structural relationship applies also to the corresponding subfamilies of PLP-dependent enzymes. We thus propose that the CoA-subfamily (including ALAS) and the aminotransferase subfamily II (including GSAM) are evolutionarily closely related and that ALAS may thus have evolved from GSAM.
    • Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction

      Fang, Kechi; Zhao, Hansheng; Sun, Changyue; Lam, Carolyn M C; Chang, Suhua; Zhang, Kunlin; Panda, Gurudutta; Godinho, Miguel; Martins dos Santos, Vítor A P; Wang, Jing (2011-05-25)
      Abstract Background Burkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets. Results We reconstructed the genome-scale metabolic network of B. cenocepacia J2315. An iterative reconstruction process led to the establishment of a robust model, iKF1028, which accounts for 1,028 genes, 859 internal reactions, and 834 metabolites. The model iKF1028 captures important metabolic capabilities of B. cenocepacia J2315 with a particular focus on the biosyntheses of key metabolic virulence factors to assist in understanding the mechanism of disease infection and identifying potential drug targets. The model was tested through BIOLOG assays. Based on the model, the genome annotation of B. cenocepacia J2315 was refined and 24 genes were properly re-annotated. Gene and enzyme essentiality were analyzed to provide further insights into the genome function and architecture. A total of 45 essential enzymes were identified as potential therapeutic targets. Conclusions As the first genome-scale metabolic network of B. cenocepacia J2315, iKF1028 allows a systematic study of the metabolic properties of B. cenocepacia and its key metabolic virulence factors affecting the CF community. The model can be used as a discovery tool to design novel drugs against diseases caused by this notorious pathogen.
    • Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction.

      Fang, Kechi; Zhao, Hansheng; Sun, Changyue; Lam, Carolyn M C; Chang, Suhua; Zhang, Kunlin; Panda, Gurudutta; Godinho, Miguel; Martins dos Santos, Vítor A P; Wang, Jing (2011)
      Burkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF) or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets.
    • Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

      Busso, Didier; Peleg, Yoav; Heidebrecht, Tatjana; Romier, Christophe; Jacobovitch, Yossi; Dantes, Ada; Salim, Loubna; Troesch, Edouard; Schuetz, Anja; Heinemann, Udo; et al. (2011-08)
      Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
    • Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath.

      Myllykoski, Matti; Kursula, Petri (2010)
      2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain.