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  • 51.
    Sjöberg, Britt-Marie
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Torrents, Eduard
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Shift in Ribonucleotide Reductase Gene Expression in Pseudomonas aeruginosa during Infection2011In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, no 7, p. 2663-2669Article in journal (Refereed)
    Abstract [en]

    The roles of different ribonucleotide reductases (RNRs) in bacterial pathogenesis have not been studied systematically. In this work we analyzed the importance of the different Pseudomonas aeruginosa RNRs in pathogenesis using the Drosophila melanogaster host-pathogen interaction model. P. aeruginosa codes for three different RNRs with different environmental requirements. Class II and III RNR chromosomal mutants exhibited reduced virulence in this model. Translational reporter fusions of RNR gene nrdA, nrdJ, or nrdD to the green fluorescent protein were constructed to measure the expression of each class during the infection process. Analysis of the P. aeruginosa infection by flow cytometry revealed increased expression of nrdJ and nrdD and decreased nrdA expression during the infection process. Expression of each RNR class fits with the pathogenicities of the chromosomal deletion mutants. An extended understanding of the pathogenicity and physiology of P. aeruginosa will be important for the development of novel drugs against infections in cystic fibrosis patients.

  • 52.
    Srinivas, Vivek
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lebrette, Hugo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kutin, Yuri
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerche, Michael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eirich, Jürgen
    Branca, Rui M. M.
    Cox, Nicholas
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stanford University School of Medicine, USA.
    Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens2018In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 563, p. 416-420Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5,6,7. Here we describe a group of RNR proteins in Mollicutes—including Mycoplasma pathogens—that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR—some of which are developing resistance to antibiotics—are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

  • 53.
    Tholander, Fredrik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Karolinska Institutet, Sweden.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Discovery of antimicrobial ribonucleotide reductase inhibitors by screening in microwell format2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 25, p. 9798-9803Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes reduction of the four different ribonucleotides to their corresponding deoxyribonucleotides and is the rate-limiting enzyme in DNA synthesis. RNR is a well-established target for the antiproliferative drugs Gemzar and Hydrea, for antisense therapy, and in combination chemotherapies. Surprisingly, few novel drugs that target RNR have emerged, partly because RNR activity assays are laboratory-intense and exclude high-throughput methodologies. Here, we present a previously undescribed PCR-based assay for RNR activity measurements in microplate format. We validated the approach by screening a diverse library of 1,364 compounds for inhibitors of class I RNR from the opportunistic pathogen Pseudomonas aeruginosa, and we identified 27 inhibitors with IC50 values from similar to 200 nM to 30 mu M. Interestingly, a majority of the identified inhibitors have been found inactive in human cell lines as well as in anticancer and in vivo tumor tests as reported by the PubChem BioAssay database. Four of the RNR inhibitors inhibited growth of P. aeruginosa, and two were also found to affect the transcription of RNR genes and to decrease the cellular deoxyribonucleotide pools. This unique PCR-based assay works with any RNR enzyme and any substrate nucleotide, and thus opens the door to high-throughput screening for RNR inhibitors in drug discovery.

  • 54.
    Thureborn, Petter
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Södertorn University College, Sweden.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Södertorn University College, Sweden.
    Plathan, Josefin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony M.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. University of Canterbury, New Zealand.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöling, Sara
    A Metagenomics Transect into the Deepest Point of the Baltic Sea Reveals Clear Stratification of Microbial Functional Capacities2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e74983-Article in journal (Refereed)
    Abstract [en]

    The Baltic Sea is characterized by hyposaline surface waters, hypoxic and anoxic deep waters and sediments. These conditions, which in turn lead to a steep oxygen gradient, are particularly evident at Landsort Deep in the Baltic Proper. Given these substantial differences in environmental parameters at Landsort Deep, we performed a metagenomic census spanning surface to sediment to establish whether the microbial communities at this site are as stratified as the physical environment. We report strong stratification across a depth transect for both functional capacity and taxonomic affiliation, with functional capacity corresponding most closely to key environmental parameters of oxygen, salinity and temperature. We report similarities in functional capacity between the hypoxic community and hadal zone communities, underscoring the substantial degree of eutrophication in the Baltic Proper. Reconstruction of the nitrogen cycle at Landsort deep shows potential for syntrophy between archaeal ammonium oxidizers and bacterial denitrification at anoxic depths, while anaerobic ammonium oxidation genes are absent, despite substantial ammonium levels below the chemocline. Our census also reveals enrichment in genetic prerequisites for a copiotrophic lifestyle and resistance mechanisms reflecting adaptation to prevalent eutrophic conditions and the accumulation of environmental pollutants resulting from ongoing anthropogenic pressures in the Baltic Sea.

  • 55.
    Torrents, Eduard
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poplawski, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Two proteins mediate class II ribonucleotide reductase activity in Pseudomonas aeruginosa: expression and transcriptional analysis of the aerobic enzymes.2005In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 17, p. 16571-8Article in journal (Other academic)
    Abstract [en]

    The opportunistic human pathogen Pseudomonas aeruginosa is one of a few microorganisms that code for three different classes (I, II, and III) of the enzyme ribonucleotide reductase (RNR). Class II RNR of P. aeruginosa differs from all hitherto known class II enzymes by being encoded by two consecutive open reading frames denoted nrdJa and nrdJb and separated by 16 bp. Split nrdJ genes were also found in the few other gamma-proteobacteria that code for a class II RNR. Interestingly, the two genes encoding the split nrdJ in P. aeruginosa were co-transcribed, and both proteins were expressed. Exponentially growing aerobic cultures were predominantly expressing the class I RNR (encoded by the nrdAB operon) compared with the class II RNR (encoded by the nrdJab operon). Upon entry to stationary phase, the relative amount of nrdJa transcript increased about 6-7-fold concomitant with a 6-fold decrease in the relative amount of nrdA transcript. Hydroxyurea treatment known to knock out the activity of class I RNR caused strict growth inhibition of P. aeruginosa unless 5'-deoxyadenosylcobalamin, a cofactor specifically required for activity of class II RNRs, was added to the rich medium. Rescue of the hydroxyurea-treated cells in the presence of the vitamin B12 cofactor strongly implies that P. aeruginosa produces a functionally active NrdJ protein. Biochemical studies showed for the first time that presence of both NrdJa and NrdJb subunits were absolutely essential for enzyme activity. Based on combined genetic and biochemical results, we suggest that the two-component class II RNR in P. aeruginosa is primarily used for DNA repair and/or possibly DNA replication at low oxygen tension.

  • 56.
    Torrents, Eduard
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Biglino, Daniele
    Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical.2005In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 102, no 50, p. 17946-51Article in journal (Other academic)
    Abstract [en]

    Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60 degrees rotational angle of C(alpha)-(C(beta)-C(1))-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens.

  • 57. Torrents, Eduard
    et al.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The Ribonucleotide Reductase Family: Genetics and Genomics2008In: Ribonucleotide Reductase, Nova Science Publishers Inc , 2008Chapter in book (Other (popular science, discussion, etc.))
  • 58. Torrents, Eduard
    et al.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Antibacterial activity of radical scavengers against reductase from Bacillus anthracis2010In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 391, no 2/3, p. 229-234Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis is a severe mammalian pathogen. The deoxyribonucleotides necessary for DNA replication and repair are provided via the ribonucleotide reductase (RNR) enzyme. RNR is also important for spore germination and cell proliferation upon infection. We show that the expression of B. anthracis class Ib RNR responds to the environment that the pathogen encounters upon infection. We also show that several anti-proliferative agents (radical scavengers) specifically inhibit the B. anthracis RNR. Owing to the importance of RNR in the pathogenic infection process, our results highlight a promising potential to inhibit the growth of B. anthracis early during infection.

  • 59.
    Torrents, Eduard
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Westman, MariAnn
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ribonucleotide reductase modularity: Atypical duplication of the ATP-cone domain in Pseudomonas aeruginosa.2006In: J Biol Chem, ISSN 0021-9258, Vol. 281, no 35, p. 25287-96Article in journal (Other academic)
    Abstract [en]

    The opportunistic pathogen Pseudomonas aeruginosa, which causes serious nosocomial infections, is a gamma-proteobacterium that can live in many different environments. Interestingly P. aeruginosa encodes three ribonucleotide reductases (RNRs) that all differ from other well known RNRs. The RNR enzymes are central for de novo synthesis of deoxyribonucleotides and essential to all living cells. The RNR of this study (class Ia) is a complex of the NrdA protein harboring the active site and the allosteric sites and the NrdB protein harboring a tyrosyl radical necessary to initiate catalysis. P. aeruginosa NrdA contains an atypical duplication of the N-terminal ATP-cone, an allosteric domain that can bind either ATP or dATP and regulates the overall enzyme activity. Here we characterized the wild type NrdA and two truncated NrdA variants with precise N-terminal deletions. The N-terminal ATP-cone (ATP-c1) is allosterically functional, whereas the internal ATP-cone lacks allosteric activity. The P. aeruginosa NrdB is also atypical with an unusually short lived tyrosyl radical, which is efficiently regenerated in presence of oxygen as the iron ions remain tightly bound to the protein. The P. aeruginosa wild type NrdA and NrdB proteins form an extraordinarily tight complex with a suggested alpha4beta4 composition. An alpha2beta2 composition is suggested for the complex of truncated NrdA (lacking ATP-c1) and wild type NrdB. Duplication or triplication of the ATP-cone is found in some other bacterial class Ia RNRs. We suggest that protein modularity built on the common catalytic core of all RNRs plays an important role in class diversification within the RNR family.

12 51 - 59 of 59
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