41 results on '"Stieglitz B"'
Search Results
2. A Pseudomonas putida capable of stereoselective hydrolysis of nitriles
- Author
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Fallon, R. D., Stieglitz, B., and Turner Jr., I.
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- 1997
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3. Patents and literature
- Author
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Linhardt, Robert J., Cavazza C., Chibata I., Tosa T., Mori T., Fujimura M., Ensley B. D., Fujiwara M., Fujiwara A., Miyamoto C., Goldberg I., Stieglitz B., Goodhue T., Kydd G. C., Foster H., McCombs C. A., Higgins I. J., Hill F. F., Schindler J., Schmid R., Preuss W., Struve A., Hsieh J. H., Barer S. J., Maxwell P. C., Imada Y., Osozawa T., Morimoto Y., Kinoshita M., Knight J. C., Wovcha M. G., Kominek L. A., Wolf H. J., Krasnobajew V., Kula M. R., Hummel W., Schutte H., Leuchtenberger W., Kurtzman P., Bothast R. J., VanCauwenberge J. E., Manecke G., Klussman U., Marsheck W. J., Jiu J., Wang P. T., McCullough J. E., Mitchell T. G., Barnes A. G., Jackson J. S., Bevan P. C., Oki T., Yoshimoto A., Matsuzawa Y., Inui T., Takeuchi T., Umezawa H., Kouno K., Sawada, Haruji, Taguchi H., Seely R. J., Sih C. J., Sonoyama T., Kageyama B., Honjo T., Teichmuller G., Rabe J., Henkel H., Terahara A., Tanaka M., Teraham A., Turner J. R., Krupinski V. M., Fukuda D. S., Baltz R. H., Udvardy N., Cserey P., Bartho I., Hantos G., Trinn M., Vida Z., Szejtli J., Stadler E., Szoke A., Habon I., Bal E., Czurda M. E., Weber A., Kennecke M., Müller R., Biggs C. B., Zeikus J. G., and Lamed R. J.
- Published
- 1986
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4. Crystal structure of the catalytic core of E3 ligase HOIP
- Author
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Stieglitz, B., primary, Rana, R.R., additional, Koliopoulos, M.G., additional, Morris-Davies, A.C., additional, Christodoulou, E., additional, Howell, S., additional, Brown, N.R., additional, and Rittinger, K., additional
- Published
- 2013
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5. Structure of an active ligase (HOIP)/ubiquitin transfer complex
- Author
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Rana, R.R., primary, Stieglitz, B., additional, Koliopoulos, M.G., additional, Morris-Davies, A.C., additional, Christodoulou, E., additional, Howell, S., additional, Brown, N.R., additional, and Rittinger, K., additional
- Published
- 2013
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6. Structure of an active ligase (HOIP-H889A)/ubiquitin transfer complex
- Author
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Rana, R.R., primary, Stieglitz, B., additional, Koliopoulos, M.G., additional, Morris-Davies, A.C., additional, Christodoulou, E., additional, Howell, S., additional, Brown, N.R., additional, and Rittinger, K., additional
- Published
- 2013
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7. Crystal structure of the PH domain of SHARPIN
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Stieglitz, B., primary, Haire, L.F., additional, Dikic, I., additional, and Rittinger, K., additional
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- 2012
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8. Crystal Structure of NORE1A in Complex with RAS
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Stieglitz, B., primary, Bee, C., additional, Schwarz, D., additional, Yildiz, O., additional, Moshnikova, A., additional, Khokhlatchev, A., additional, and Herrmann, C., additional
- Published
- 2008
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9. Crystal structure of the Clostridium limosum C3 exoenzyme
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Vogelsgesang, M., primary, Stieglitz, B., additional, Herrmann, C., additional, Pautsch, A., additional, and Aktories, K., additional
- Published
- 2008
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10. Identification of Microbial and Rat Metabolites of Triflusulfuron Methyl, A New Sugar Beet Herbicide
- Author
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Dietrich, Robert F., primary, Reiser, Robert W., additional, and Stieglitz, B., additional
- Published
- 1995
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11. Patents and literature.
- Author
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Linhardt, Robert, Cavazza, C., Chibata, I., Tosa, T., Mori, T., Fujimura, M., Ensley, B., Fujiwara, M., Fujiwara, A., Miyamoto, C., Goldberg, I., Stieglitz, B., Goodhue, T., Kydd, G., Foster, H., McCombs, C., Higgins, I., Hill, F., Schindler, J., and Schmid, R.
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- 1987
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12. Mechanized systems for media dispensing, inoculation, and replication of microorganisms.
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Stieglitz, B. and DeFelice, C. P.
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- 1986
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13. Discovery and Development of a Commercial Synthesis of Azafenidin
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Shapiro, R., DiCosimo, R., Hennessey, S. M., Stieglitz, B., Campopiano, O., and Chiang, G. C.
- Abstract
A commercial synthesis of the DuPont herbicide azafenidin is described. Discovery of a novel synthesis of the triazolinone ring system and a practical, environmentally benign process to 5-cyanovaleramide were critical breakthroughs in enabling azafenidin to be manufactured at an acceptable cost. The process began with the selective hydrolysis of DuPont's nylon intermediate, adiponitrile, to 5-cyanovaleramide. This was converted via Hofmann rearrangement and Pinner-type cyclization to afford the key amidine carboxylate intermediate containing both carbon atoms of the triazolinone ring. The preservation of all six carbon atoms of adiponitrile set up a 2 + 3 cyclocondensation with arylhydrazines, which replaced a costly 4 + 1 cyclocondensation of an amidrazone with phosgene or a phosgene surrogate used in the original route. This new triazolinone process was optimized to afford the commercial product in a highly efficient and economical fashion.
- Published
- 2001
14. Distribution of the Isopropylmalate Pathway to Leucine Among Diverse Bacteria
- Author
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Stieglitz, B. I. and Calvo, J. M.
- Abstract
α-Isopropylmalate synthase and β-isopropylmalate dehydrogenase activities were detected in extracts of the following organisms: ChromatiumD, Rhodopseudomonas spheroides, HydrogenomonasH16, Pseudomonas aeruginosa, Pseudomonas fluorescens, Vibrio extorquens, Rhizobium japonicum, Alcaligenes viscolactis, Escherichia coliB, Proteus vulgaris, Aerobacter aerogenes, Salmonella typhimurium, Micrococcussp., Micrococcus lysodeikticus, Bacillus polymyxa, Bacillus subtilis, and Nocardia opaca. The α-isopropylmalate synthase activity in these extracts was inhibited by low concentrations of l-leucine. Taken together with other data, these results suggest that the isopropylmalate pathway is widespread among organisms that can synthesize leucine.
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- 1974
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15. Effect of 4-Azaleucine upon Leucine Metabolism in Salmonella typhimurium
- Author
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Stieglitz, B. and Calvo, J. M.
- Abstract
dl-4-Azaleucine (5 × 3−3m) added to exponentially growing cells of Salmonella typhimuriumresulted in an abrupt cessation of growth lasting 4 to 8 hr followed by a resumption of division. The transitory nature of inhibition was not due to the instability or modification of the analogue or to a derepression of leucine-forming enzymes. Of many compounds tested, leucine served most efficiently to reverse 4-azaleucine-induced inhibition. Inhibition of growth can be explained by the fact that 4-azaleucine inhibits α-isopropylmalate synthase, the first enzyme unique to leucine biosynthesis. The analogue was a poor inhibitor of both the transamination of α-ketoisocaproate to leucine and the charging of leucine to transfer ribonucleic acid. With a leucine auxotroph starved for leucine, the analogue was incorporated into protein specifically in place of leucine. Such incorporation was accompanied by the death of almost all of the cells.
- Published
- 1971
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16. Methanol Metabolism in Pseudomonad C
- Author
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Stieglitz, B. and Mateles, R. I.
- Abstract
Cell suspensions of pseudomonad C, a bacterium capable of growth on methanol as sole carbon source, were able to oxidize methanol, formaldehyde, and formate, although the rates of oxidation for the latter two compounds were much slower. The latter compounds also could not serve as sole carbon sources. Through the use of labeled compounds, it was shown that in the presence of methanol, formaldehyde, formate, and bicarbonate were incorporated into trichloroacetic acid-precipitable material. Hexose phosphate synthetase activity was found, indicating the assimilation of methanol via an allulose pathway. No hydroxypyruvate reductase activity was found, nor was any complex membrane structure observed. Such a combination of characteristics has been observed in an obligate methylotroph (PseudomonasW1), but pseudomonad C can utilize a variety of non-methyl substrates.
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- 1973
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17. Leucyl-Transfer Ribonucleic Acid Synthetase from a Wild-Type and Temperature-Sensitive Mutant of Salmonella typhimurium1
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Mikulka, T. W., Stieglitz, B. I., and Calvo, J. M.
- Abstract
Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following Kmvalues: leucine, 1.1 × 10−5m; adenosine triphosphate, 6.5 × 10−4m; tRNAILeu, 4.1 × 10−8m; tRNAIILeu, 4.3 × 10−8m; tRNAIIILeu, 5.3 × 10−8m; and tRNAIVLeu, 2.9 × 10−8m. The tRNALeufractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min−1. Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.
- Published
- 1972
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18. Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase
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Goldberg, I, primary, Lonberg-Holm, K, additional, Bagley, E A, additional, and Stieglitz, B, additional
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- 1983
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19. Novel microbial screen for detection of 1,4-butanediol, ethylene glycol, and adipic acid
- Author
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Stieglitz, B, primary and Weimer, P J, additional
- Published
- 1985
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20. N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling.
- Author
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Kliza KW, Song W, Pinzuti I, Schaubeck S, Kunzelmann S, Kuntin D, Fornili A, Pandini A, Hofmann K, Garnett JA, Stieglitz B, and Husnjak K
- Abstract
Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death., (© 2024. The Author(s).)
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- 2024
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21. Structural basis for ubiquitylation by HOIL-1.
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Wu Q, Koliopoulos MG, Rittinger K, and Stieglitz B
- Abstract
The linear ubiquitin chain assembly complex synthesises linear Ub chains which constitute a binding and activation platform for components of the TNF signalling pathway. One of the components of LUBAC is the ubiquitin ligase HOIL-1 which has been shown to generate oxyester linkages on several proteins and on linear polysaccharides. We show that HOIL-1 activity requires linear tetra-Ub binding which enables HOIL-1 to mono-ubiquitylate linear Ub chains and polysaccharides. Furthermore, we describe the crystal structure of a C-terminal tandem domain construct of HOIL-1 comprising the IBR and RING2 domains. Interestingly, the structure reveals a unique bi-nuclear Zn-cluster which substitutes the second zinc finger of the canonical RING2 fold. We identify the C-terminal histidine of this bi-nuclear Zn-cluster as the catalytic base required for the ubiquitylation activity of HOIL-1. Our study suggests that the unique zinc-coordinating architecture of RING2 provides a binding platform for ubiquitylation targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Koliopoulos, Rittinger and Stieglitz.)
- Published
- 2023
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22. HHARI in motion reveals an unexpected substrate recognition site for RBR ligases.
- Author
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Stieglitz B
- Subjects
- Models, Molecular, Protein Conformation, Ubiquitin metabolism, Ubiquitin-Conjugating Enzymes metabolism, Ligases metabolism, Ubiquitin-Protein Ligases chemistry
- Abstract
Capturing the enzymatic activity of RBR ligases in molecular detail is challenging due to their inherent dynamic behavior. In this issue of Structure, Reiter and colleagues tackle this problem using a multidisciplinary approach. They show that activation of the ubiquitin ligase HHARI by phosphorylation induces a major conformational rearrangement, which reveals an unexpected substrate binding site., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)
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- 2022
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23. Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development.
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Green JL, Wu Y, Encheva V, Lasonder E, Prommaban A, Kunzelmann S, Christodoulou E, Grainger M, Truongvan N, Bothe S, Sharma V, Song W, Pinzuti I, Uthaipibull C, Srichairatanakool S, Birault V, Langsley G, Schindelin H, Stieglitz B, Snijders AP, and Holder AA
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- Humans, Plasmodium falciparum genetics, Protozoan Proteins genetics, Ubiquitin genetics, Merozoites metabolism, Plasmodium falciparum metabolism, Protozoan Proteins metabolism, Ubiquitin metabolism, Ubiquitination
- Abstract
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites., Competing Interests: The authors have declared that no competing interests exist.
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- 2020
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24. Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP.
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Burlina F, Abdel-Aal AM, Raz R, Pinzuti I, Papageorgiou G, Li J, Antrobus R, Martin SR, Kunzelmann S, Stieglitz B, and Offer J
- Abstract
The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMO
CoZi -Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step requiring different LUBAC components., Competing Interests: Competing interests: The authors declare no competing interests.- Published
- 2019
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25. Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.
- Author
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Kliza K, Taumer C, Pinzuti I, Franz-Wachtel M, Kunzelmann S, Stieglitz B, Macek B, and Husnjak K
- Subjects
- HEK293 Cells, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins, NF-kappa B metabolism, Polyubiquitin genetics, Protein Processing, Post-Translational, Signal Transduction, Tumor Necrosis Factor-alpha, Ubiquitin-Protein Ligase Complexes metabolism, Endopeptidases metabolism, Polyubiquitin metabolism, TNF Receptor-Associated Factor 6 metabolism, Ubiquitin-Protein Ligases metabolism, Ubiquitination
- Abstract
Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-REx HEK293T cells and validated several newly identified linear polyubiquitin targets. We demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for NFκB signaling.
- Published
- 2017
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26. Determination of the pK a of the N-terminal amino group of ubiquitin by NMR.
- Author
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Oregioni A, Stieglitz B, Kelly G, Rittinger K, and Frenkiel T
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- Catalytic Domain, Humans, Hydrogen-Ion Concentration, Kinetics, Magnetic Resonance Spectroscopy, Models, Molecular, Protein Conformation, Ubiquitin metabolism, Ubiquitination, Protein Interaction Domains and Motifs, Ubiquitin chemistry
- Abstract
Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pK
a of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains.- Published
- 2017
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27. Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.
- Author
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Dove KK, Stieglitz B, Duncan ED, Rittinger K, and Klevit RE
- Subjects
- Binding Sites, Catalytic Domain, Humans, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Polycomb Repressive Complex 1 chemistry, Polycomb Repressive Complex 1 metabolism, Protein Binding, Protein Conformation, Protein Transport, Tumor Suppressor Proteins chemistry, Tumor Suppressor Proteins metabolism, Ubiquitin chemistry, Ubiquitin Thiolesterase chemistry, Ubiquitin Thiolesterase metabolism, Ubiquitin-Conjugating Enzymes chemistry, Ubiquitin-Conjugating Enzymes metabolism, Ubiquitin-Protein Ligases chemistry, Ubiquitination, Ubiquitin metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs., (© 2016 The Authors.)
- Published
- 2016
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28. Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP.
- Author
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Stieglitz B, Rana RR, Koliopoulos MG, Morris-Davies AC, Schaeffer V, Christodoulou E, Howell S, Brown NR, Dikic I, and Rittinger K
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- Apoproteins chemistry, Apoproteins metabolism, Catalytic Domain, Crystallography, X-Ray, HeLa Cells, Humans, Models, Molecular, Protein Conformation, Substrate Specificity, Ubiquitin chemistry, Ubiquitin metabolism, Ubiquitin-Protein Ligases chemistry, Ubiquitin-Protein Ligases metabolism
- Abstract
Linear ubiquitin chains are important regulators of cellular signalling pathways that control innate immunity and inflammation through nuclear factor (NF)-κB activation and protection against tumour necrosis factor-α-induced apoptosis. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multisubunit E3 ligase. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or 'donor' ubiquitin. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The carboxy-terminal portion of HOIP adopts a novel fold that, together with a zinc-finger, forms a ubiquitin-binding platform that orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3∼ubiquitin thioester. The C-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine, providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-κB pathway in vivo, and they explain the determinants of linear ubiquitin chain specificity by LUBAC.
- Published
- 2013
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29. LUBAC synthesizes linear ubiquitin chains via a thioester intermediate.
- Author
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Stieglitz B, Morris-Davies AC, Koliopoulos MG, Christodoulou E, and Rittinger K
- Subjects
- Animals, Catalytic Domain, Cattle, Esters chemistry, Polyubiquitin chemistry, Protein Structure, Quaternary, Protein Structure, Tertiary, Protein Subunits, Sulfhydryl Compounds chemistry, Polyubiquitin biosynthesis, Ubiquitin-Protein Ligases chemistry
- Abstract
The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP--the catalytic subunit of LUBAC--is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.
- Published
- 2012
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30. Crystallization of SHARPIN using an automated two-dimensional grid screen for optimization.
- Author
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Stieglitz B, Rittinger K, and Haire LF
- Subjects
- Automation, Laboratory, Crystallization, Crystallography, X-Ray, Humans, Carrier Proteins chemistry, Ubiquitins chemistry
- Abstract
An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli, purified and crystallized. Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen. The crystals belonged to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 61.55, c = 222.81 Å. Complete data sets were collected from native and selenomethionine-substituted protein crystals at 100 K to 2.6 and 2.0 Å resolution, respectively.
- Published
- 2012
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31. Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold.
- Author
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Stieglitz B, Haire LF, Dikic I, and Rittinger K
- Subjects
- Blood Proteins chemistry, Crystallography, X-Ray, Humans, Phosphoproteins chemistry, Protein Structure, Quaternary, Protein Structure, Tertiary, Structure-Activity Relationship, Zinc Fingers, Nerve Tissue Proteins chemistry, Protein Folding, Protein Multimerization, Protein Subunits chemistry, Ubiquitin-Protein Ligases chemistry
- Abstract
SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.
- Published
- 2012
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32. An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation.
- Author
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Wilhelm C, Hirota K, Stieglitz B, Van Snick J, Tolaini M, Lahl K, Sparwasser T, Helmby H, and Stockinger B
- Subjects
- Animals, Antibodies, Blocking administration & dosage, Cells, Cultured, Cytokines immunology, Genes, Reporter genetics, Immunity, Innate, Interleukin-9 genetics, Interleukin-9 immunology, Lung, Lymphocyte Activation drug effects, Lymphocytes immunology, Lymphocytes pathology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Papain administration & dosage, Paracrine Communication, Pneumonia chemically induced, Th2 Cells drug effects, Cytokines metabolism, Interleukin-9 metabolism, Lymphocytes metabolism, Pneumonia immunology, Th2 Cells immunology
- Abstract
Interleukin 9 (IL-9) is a cytokine linked to lung inflammation, but its cellular origin and function remain unclear. Here we describe a reporter mouse strain designed to map the fate of cells that have activated IL-9. We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs). IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5. Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses.
- Published
- 2011
- Full Text
- View/download PDF
33. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis.
- Author
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Ikeda F, Deribe YL, Skånland SS, Stieglitz B, Grabbe C, Franz-Wachtel M, van Wijk SJ, Goswami P, Nagy V, Terzic J, Tokunaga F, Androulidaki A, Nakagawa T, Pasparakis M, Iwai K, Sundberg JP, Schaefer L, Rittinger K, Macek B, and Dikic I
- Subjects
- Animals, B-Lymphocytes metabolism, Carrier Proteins metabolism, Caspase 8 metabolism, Cells, Cultured, Dermatitis genetics, Dermatitis metabolism, Dermatitis pathology, Fas-Associated Death Domain Protein metabolism, Fibroblasts metabolism, HEK293 Cells, HeLa Cells, Humans, I-kappa B Kinase metabolism, Intracellular Signaling Peptides and Proteins metabolism, Macrophages metabolism, Mice, Nerve Tissue Proteins deficiency, Nerve Tissue Proteins genetics, Tumor Necrosis Factor-alpha metabolism, Tumor Necrosis Factor-alpha pharmacology, Ubiquitin-Protein Ligases metabolism, Ubiquitination, Apoptosis drug effects, NF-kappa B metabolism, Nerve Tissue Proteins metabolism, Ubiquitin metabolism, Ubiquitin-Protein Ligase Complexes metabolism
- Abstract
SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.
- Published
- 2011
- Full Text
- View/download PDF
34. Growth and tumor suppressor NORE1A is a regulatory node between Ras signaling and microtubule nucleation.
- Author
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Bee C, Moshnikova A, Mellor CD, Molloy JE, Koryakina Y, Stieglitz B, Khokhlatchev A, and Herrmann C
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Animals, Apoptosis Regulatory Proteins, Aurora Kinase A, Aurora Kinases, Cell Line, Humans, Mice, Microtubules genetics, Monomeric GTP-Binding Proteins genetics, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Tubulin genetics, Tubulin metabolism, Tumor Suppressor Proteins genetics, ras Proteins genetics, Adaptor Proteins, Signal Transducing metabolism, Microtubules metabolism, Monomeric GTP-Binding Proteins metabolism, Signal Transduction physiology, Tumor Suppressor Proteins metabolism, ras Proteins metabolism
- Abstract
NORE1A is a Ras-binding protein that belongs to a group of tumor suppressors known as the Ras association domain family. Their growth- and tumor-suppressive function is assumed to be dependent on association with the microtubule cytoskeleton. However, a detailed understanding of this interplay is still missing. Here, we show that NORE1A directly interacts with tubulin and is capable of nucleating microtubules. Strikingly, the ability to stimulate nucleation is regulated in a dual specific way either via phosphorylation of NORE1A within the Ras-binding domain by Aurora A kinase or via binding to activated Ras. We also demonstrate that NORE1A mediates a negative effect of activated Ras on microtubule nucleation. On the basis of our results, we propose a novel regulatory network composed of the tumor suppressor NORE1A, the mitotic kinase Aurora A, the small GTPase Ras, and the microtubule cytoskeleton.
- Published
- 2010
- Full Text
- View/download PDF
35. Novel type of Ras effector interaction established between tumour suppressor NORE1A and Ras switch II.
- Author
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Stieglitz B, Bee C, Schwarz D, Yildiz O, Moshnikova A, Khokhlatchev A, and Herrmann C
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Amino Acid Sequence, Animals, Apoptosis Regulatory Proteins, Binding Sites, Cell Line, Tumor, Crystallography, X-Ray, Humans, Mice, Models, Molecular, Molecular Sequence Data, Monomeric GTP-Binding Proteins genetics, Mutagenesis, Protein Interaction Domains and Motifs, Tumor Suppressor Proteins chemistry, Tumor Suppressor Proteins metabolism, Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing metabolism, Monomeric GTP-Binding Proteins chemistry, Monomeric GTP-Binding Proteins metabolism, ras Proteins metabolism
- Abstract
A class of putative Ras effectors called Ras association domain family (RASSF) represents non-enzymatic adaptors that were shown to be important in tumour suppression. RASSF5, a member of this family, exists in two splice variants known as NORE1A and RAPL. Both of them are involved in distinct cellular pathways triggered by Ras and Rap, respectively. Here we describe the crystal structure of Ras in complex with the Ras binding domain (RBD) of NORE1A/RAPL. All Ras effectors share a common topology in their RBD creating an interface with the switch I region of Ras, whereas NORE1A/RAPL RBD reveals additional structural elements forming a unique Ras switch II binding site. Consequently, the contact area of NORE1A is extended as compared with other Ras effectors. We demonstrate that the enlarged interface provides a rationale for an exceptionally long lifetime of the complex. This is a specific attribute characterizing the effector function of NORE1A/RAPL as adaptors, in contrast to classical enzymatic effectors such as Raf, RalGDS or PI3K, which are known to form highly dynamic short-lived complexes with Ras.
- Published
- 2008
- Full Text
- View/download PDF
36. Crystal structure of the Clostridium limosum C3 exoenzyme.
- Author
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Vogelsgesang M, Stieglitz B, Herrmann C, Pautsch A, and Aktories K
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Molecular Sequence Data, Protein Conformation, ADP Ribose Transferases chemistry, Botulinum Toxins chemistry, Models, Molecular
- Abstract
C3-like toxins ADP-ribosylate and inactivate Rho GTPases. Seven C3-like ADP-ribosyltransferases produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus were identified and two representatives--C3bot from C. botulinum and C3stau2 from S. aureus--were crystallized. Here we present the 1.8A structure of C. limosum C3 transferase C3lim and compare it to the structures of other family members. In contrast to the structure of apo-C3bot, the canonical ADP-ribosylating turn turn motif is observed in a primed conformation, ready for NAD binding. This suggests an impact on the binding mode of NAD and on the transferase reaction. The crystal structure explains why auto-ADP-ribosylation of C3lim at Arg41 interferes with the ADP-ribosyltransferase activity of the toxin.
- Published
- 2008
- Full Text
- View/download PDF
37. Adsorption of hydrophobin proteins at hydrophobic and hydrophilic interfaces.
- Author
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Lumsdon SO, Green J, and Stieglitz B
- Subjects
- Adsorption, Chemistry, Physical methods, Emulsions, Fatty Acids, Unsaturated chemistry, Fungal Proteins classification, Hydrophobic and Hydrophilic Interactions, Kinetics, Oils chemistry, Polytetrafluoroethylene chemistry, Powders, Surface Properties, Surface Tension, Water chemistry, Wettability, Fungal Proteins pharmacokinetics, Schizophyllum chemistry
- Abstract
The surface activity of two hydrophobin proteins, HFBII and SC3, at the solid-liquid, liquid-liquid and liquid-vapor interface has been investigated. Hydrophobins are fungal proteins that are known to adsorb and affect the physico-chemical properties of an interface. In this study, the surface activity was determined by measuring the interaction of hydrophobin molecules with various liquids, solid particles and films that are commonly used or produced in industrial processes. We found that a very low concentration of hydrophobin is required to facilitate the wet-in of hydrophobic solid particles, such as Teflon, into aqueous solutions. It is also capable of stabilizing aqueous dispersions of Kevlar nanopulp, reversing the wettability of hydrophobic films and stabilizing polyunsaturated fatty acid (PUFA) oil-in-water emulsions.
- Published
- 2005
- Full Text
- View/download PDF
38. 5-Cyanovaleramide production using immobilized Pseudomonas chlororaphis B23.
- Author
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Hann EC, Eisenberg A, Fager SK, Perkins NE, Gallagher FG, Cooper SM, Gavagan JE, Stieglitz B, Hennessey SM, and DiCosimo R
- Subjects
- Amides metabolism, Enzyme Stability, Hydro-Lyases chemistry, Hydro-Lyases metabolism, Nitriles metabolism, Amides chemistry, Biotechnology methods, Nitriles chemistry, Pseudomonas metabolism
- Abstract
A biocatalytic process for the hydration of adiponitrile to 5-cyanovaleramide has been developed which can be run to higher conversion, produces more product per weight of catalyst, and generates significantly less waste products than alternate chemical processes. The biocatalyst consists of Pseudomonas chlororaphis B23 microbial cells immobilized in calcium alginate beads. The cells contain a nitrile hydratase (EC 4.2.1.84) which catalyzes the hydration of adiponitrile to 5-cyanovaleramide with high regioselectivity, and with less than 5% selectivity to byproduct adipamide. Fifty-eight consecutive batch reactions with biocatalyst recycle were run to convert a total of 12.7 metric tons of adiponitrile to 5-cyanovaleramide. At 97% adiponitrile conversion, the yield of 5-cyanovaleramide was 13.6 metric tons (93% yield, 96% selectivity), and the total weight of 5-cyanovaleramide produced per weight of catalyst was 3150 kg/kg (dry cell weight).
- Published
- 1999
- Full Text
- View/download PDF
39. A stereoselective cobalt-containing nitrile hydratase.
- Author
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Payne MS, Wu S, Fallon RD, Tudor G, Stieglitz B, Turner IM Jr, and Nelson MJ
- Subjects
- Amino Acid Sequence, Base Sequence, Catalysis, Cloning, Molecular, Cobalt metabolism, Electron Spin Resonance Spectroscopy, Genes, Bacterial, Hydro-Lyases isolation & purification, Hydro-Lyases metabolism, Hydrogen-Ion Concentration, Molecular Sequence Data, Nitriles metabolism, Pseudomonas putida genetics, Stereoisomerism, Temperature, Cobalt chemistry, Hydro-Lyases chemistry, Pseudomonas putida enzymology
- Abstract
Nitrile hydratase from Pseudomonas putida NRRL-18668 has been purified and characterized. The purified enzyme catalyzes the hydration of 2(S)-(4'-chlorophenyl)-3-methylbutyronitrile at least fifty times faster than that of 2(R)-(4'-chlorophenyl)-3-methylbutyronitrile. This enzyme is a member of the class of nitrile hydratase that contains cobalt. Visible absorption and CD spectra suggest the cobalt exists as a non-corrin low-spin Co3+ ion in a tetragonally-distorted octahedral ligand field. Chemical reduction of the native enzyme results in a species with the EPR signature of a low-spin Co2+ complex. Like the other cobalt-containing nitrile hydratases, this enzyme is relatively stable, maintaining its activity below 35 degrees C, and it shows a broad activity optimum between pH 7.2 and 7.8. The structural genes for this enzyme have been cloned and sequenced. The deduced amino acid sequences for the alpha and beta subunits show 48-63% and 35-41% homology, respectively, to other sequenced nitrile hydratases. In particular, the cysteine residues in the alpha subunit that have been suggested to coordinate the metal ion in the iron-containing nitrile hydratases [Brennan, B. A., Cummings, J. G., Chase, D. B., Turner, I. M., Jr., & Nelson, M. J. (1996) Biochemistry 35, 10068-10077] are conserved in this enzyme, suggesting that this nitrile hydratase, like the enzyme from Rhodococcus rhodochrous J1, is a member of a newly described class of metalloenzymes with Co3+-thiolate ligation [Brennan, B. A., Alms, G., Nelson, M. J., Durney, L. T., & Scarrow, R. C. (1996) J. Am. Chem. Soc. 118, 9194-9195].
- Published
- 1997
- Full Text
- View/download PDF
40. Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus.
- Author
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Kenealy W, Zaady E, du Preez JC, Stieglitz B, and Goldberg I
- Abstract
The accumulation and excretion of fumaric acid, and to a lesser extent malic and succinic acids, by Rhizopus arrhizus occurs under aerobic conditions in a high-glucose medium containing a limiting amount of nitrogen and a neutralizing agent (CaCO(3)). An overall four-carbon dicarboxylic acid molar yield of up to 145% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 4 to 5 days. Evidence is presented that fumarate is synthesized from pyruvate via a carboxylation reaction yielding oxaloacetate, which is then converted to malate and further on to fumarate via the reductive reactions of the tricarboxylic acid cycle. The possible formation of fumarate from the normal (oxidative) operation of the tricarboxylic acid cycle was not excluded by the data. Yield, C nuclear magnetic resonance, and enzymatic activity studies were carried out in a strain of R. arrhizus which produces high levels of fumarate from glucose and carbonate. The observed high fumarate molar yield (greater than 100%) can therefore be explained in terms of the carboxylation of pyruvate and the operation of the reductive reactions of the tricarboxylic acid cycle under aerobic conditions.
- Published
- 1986
- Full Text
- View/download PDF
41. Fluorometric assay of enzymatic reactions involving acetyl Coenzyme A in aldol condensations.
- Author
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Calvo JM, Bartholomew JC, and Stieglitz BI
- Subjects
- Citrates analysis, Ethers, Fluorometry, Hot Temperature, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Resorcinols, Salmonella typhimurium enzymology, Spectrum Analysis, Time Factors, Ultraviolet Rays, Water, Coenzyme A analysis, Coumarins, Dicarboxylic Acids analysis, Ligases analysis, Malates analysis, Nucleotides
- Published
- 1969
- Full Text
- View/download PDF
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