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  • 1.
    Andersson, Jessica
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bodevin, Sabrina
    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.
    Two Active Site Asparagines Are Essential for the Reaction Mechanism of the Class III Anaerobic Ribonucleotide Reductase from Bacteriophage T42001In: The Journal of Biological Chemistry, Vol. 44, p. 40457-40463Article in journal (Refereed)
  • 2. Aurelius, Oskar
    et al.
    Johansson, Renzo
    Bågenholm, Viktoria
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tholander, Fredrik
    Balhuizen, Alexander
    Beck, Tobias
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mulliez, Etienne
    Logan, Derek T.
    The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0128199Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.

  • 3. Berggren, Gustav
    et al.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crona, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Swedish Orphan Biovitrum AB, Sweden.
    Tholander, Fredrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Compounds with capacity to quench the tyrosyl radical in Pseudomonas aeruginosa ribonucleotide reductase2019In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, no 6, p. 841-848Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) has been extensively probed as a target enzyme in the search for selective antibiotics. Here we report on the mechanism of inhibition of nine compounds, serving as representative examples of three different inhibitor classes previously identified by us to efficiently inhibit RNR. The interaction between the inhibitors and Pseudomonas aeruginosa RNR was elucidated using a combination of electron paramagnetic resonance spectroscopy and thermal shift analysis. All nine inhibitors were found to efficiently quench the tyrosyl radical present in RNR, required for catalysis. Three different mechanisms of radical quenching were identified, and shown to depend on reduction potential of the assay solution and quaternary structure of the protein complex. These results form a good foundation for further development of P. aeruginosa selective antibiotics. Moreover, this study underscores the complex nature of RNR inhibition and the need for detailed spectroscopic studies to unravel the mechanism of RNR inhibitors.

  • 4.
    Bergquist, Helen
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sigurdsson, Susannah
    Percipalle, Piergiorgio
    Nguyen, Chi-Hung
    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.
    Good, Liam
    Zain, Rula
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pkd1 DNA triplex stabilization by benzoquinoquinoxaline derivativesArticle in journal (Refereed)
  • 5.
    Crona, Mikael
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Avesson, Lotta
    Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala .
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hinas, Andrea
    Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala .
    Klose, Ralph
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Söderbom, Fredrik
    Department of Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala .
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    The two classes of ribonucleotide reductase in the social amoeba Dictystelium discoideumManuscript (preprint) (Other academic)
  • 6.
    Crona, Mikael
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Avesson, Lotta
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hinas, Andrea
    Klose, Ralph
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Söderbom, Fredrik
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 12, p. 8198-8208Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class IIRNRis most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B-12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.

  • 7.
    Crona, Mikael
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Torrents, Eduard
    Cellular Biotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain.
    Hofer, Anders
    Department of Medical Biochemistry & Biophysics, Umeå University.
    Furrer, Ernst
    Tomter, Ane
    Department of Molecular Biosciences, University of Oslo, Norway.
    Rohr, Åsmund
    Andersson, Kristoffer
    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.
    NrdH-redoxin mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from Bacillus anthracisManuscript (preprint) (Other academic)
  • 8.
    Crona, Mikael
    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.
    Rohr, Asmund K.
    Hofer, Anders
    Furrer, Ernst
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Tomter, Ane B.
    Andersson, K. Kristoffer
    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.
    NrdH-Redoxin Protein Mediates High Enzyme Activity in Manganese-reconstituted Ribonucleotide Reductase from Bacillus anthracis2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 38, p. 33053-33060Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

  • 9. Gustafsson, Tomas N.
    et al.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lu, Jun
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holmgren, Arne
    Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 47Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.

  • 10. Kasrayan, Alex
    et al.
    Persson, Annika L
    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 Conserved Active Site Asparagine in Class I Ribonucleotide Reductase Is Essential for Catalysis2002In: The Journal of Biological Chemistry, Vol. 277, no 8, p. 5749-5755Article in journal (Refereed)
  • 11.
    Källman, Annika M.
    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.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    ADAR2 A to I editing: site selectivity and editing efficiency are separate events2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 16, p. 4874-4881Article in journal (Refereed)
    Abstract [en]

    ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double‐stranded. Site‐selective A→I editing has been detected at specific sites within  a few tructured pre‐mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre‐mRNA of the glutamate receptor subunit B (GluR‐B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double‐stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.                 

  • 12.
    Loderer, Christoph
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jonna, Venkateswara Rao
    Crona, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rozman Grinberg, Inna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hofer, Anders
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 46, p. 19044-19054Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.

  • 13. Matsuoka, Atsuko
    et al.
    Lundin, Cecilia
    Johansson, Fredrik
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Fukuhara, Kiyoshi
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Jenssen, Dag
    Önfelt, Agneta
    Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition.2004In: Mutat Res, ISSN 0027-5107, Vol. 547, no 1-2, p. 101-7Article in journal (Other academic)
    Abstract [en]

    We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy-trans-stilbene, 3-hydroxy-trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy-trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy-trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.

  • 14. Oliw, Ernst H.
    et al.
    Jerneren, Fredrik
    Hoffmann, Inga
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Garscha, Ulrike
    Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms2011In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1811, no 3, p. 138-147Article in journal (Refereed)
    Abstract [en]

    Manganese lipoxygenase (MnLOX) oxidizes (11R)-hydroperoxylinolenic acid (11R-HpOTrE) to a peroxyl radical. Our aim was to compare the enzymatic oxidation of 11R-HpOTrE and octadecenoic acids with LOO-H and allylic C-H bond dissociation enthalpies of similar to 88 and similar to 87 kcal/mol. Mn(III)LOX oxidized (11Z)-, (12Z)-, and (13Z)-18:1 to hydroperoxides with R configuration, but this occurred at insignificant rates (<1%) compared to 11R-HpOTrE. We next examined whether transitional metals could mimic this oxidation. Ce(4+) and Mn(3+) transformed 11R-HpOTrE to hydroperoxides at C-9 and C-13 via oxidation to a peroxyl radical at C-11, whereas Fe(3+) was a poor catalyst. Our results suggest that MnLOX oxidizes bis-allylic hydroperoxides to peroxyl radicals in analogy with Ce(4+) and Mn(3+). The enzymatic oxidation likely occurs by proton-coupled electron transfer of the electron from the hydroperoxide anion to Mn(III) and H(+) to the catalytic base, Mn(III) OH(-). Hydroperoxides abolish the kinetic lag times of MnLOX and FeLOX by oxidation of their metal centers, but 11R-HpOTrE was isomerized by MnLOX to (13R)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13R-HpOTrE) with a kinetic lag time. This lag time could be explained by two competing transformations, dehydration of 11R-HpOTrE to 11-ketolinolenic acid and oxidation of 11R-HpOTrE to peroxyl radical; the reaction rate then increases as 13R-HpOTrE oxidizes MnLOX with subsequent formation of two epoxyalcohols. We conclude that oxidation of octadecenoic acids and bis-allylic hydroperoxides occurs by different mechanisms, which likely reflect the nature of the hydrogen bonds, steric factors, and the redox potential of the Mn(III) center.

  • 15. Rocca, Ignasi
    et al.
    Torrents, Eduard
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gibert, Isidre
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    NrdI Essentiality for Class Ib Ribonucleotide Reduction in Streptococcus pyogenes2008In: Journal of Bacteriology, p. 4849-4858Article in journal (Refereed)
  • 16.
    Rozman Grinberg, Inna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hasan, Mahmudul
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Lund University, Sweden.
    Crona, Mikael
    Jonna, Venkateswara Rao
    Loderer, Chrishtoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Markova, Natalia
    Borovok, Ilya
    Berggren, Gustav
    Hofer, Anders
    Logan, Derek T.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Novel ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit2018In: eLIFE, E-ISSN 2050-084X, Vol. 7, article id e31529Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 A crystal structure. We also present evidence for an (MnMnIV)-Mn-III metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.

  • 17.
    Rozman Grinberg, Inna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crona, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Swedish Orphan Biovitrum AB, Sweden.
    Berggren, Gustav
    Hofer, Anders
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 41, p. 15889-15900Article in journal (Refereed)
    Abstract [en]

    Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyze reduction of ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is a unique fusion protein with N-terminal addons of a glutaredoxin (Grx) domain followed by an ATP-binding domain, the ATP cone. Grx, usually encoded separately from the RNR operon, is a known RNR reductant. We show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and, interestingly, also via a monothiol mechanism, although less efficiently. To our knowledge, a Grx that uses both of these two reaction mechanisms has not previously been observed with a native substrate. The ATP cone is in most RNRs an N-terminal domain of the catalytic subunit. It is an allosteric on/off switch promoting ribonucleotide reduction in the presence of ATP and inhibiting RNR activity in the presence of dATP. We found that dATP bound to the ATP cone of F. ignava NrdB promotes formation of tetramers that cannot form active complexes with NrdA. The ATP cone bound two dATP molecules but only one ATP molecule. F. ignava NrdB contains the recently identified radical-generating cofactor Mn-III/Mn-IV. We show that NrdA from F. ignava can form a catalytically competent RNR with the Mn-III/Mn-IV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis. In conclusion, F. ignava NrdB is fused with a Grx functioning as an RNR reductant and an ATP cone serving as an on/off switch.

  • 18.
    Sahlin, Margareta
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Cho, Kyung-Bin
    Department of Biochemistry and Biophysics.
    Pötsch, Stephan
    Department of Biochemistry and Biophysics.
    Lytton, Simon D
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Huque, Yasmin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gunther, Michael R
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Mason, Ronald P
    Gräslund, Astrid
    Department of Biochemistry and Biophysics.
    Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli.2002In: J Biol Inorg Chem, ISSN 0949-8257, Vol. 7, no 1-2, p. 74-82Article in journal (Other academic)
    Abstract [en]

    Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.

  • 19.
    Saleh, Aljona
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Edlund, Per-Olof
    Gustafsson, Tomas N.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Granelli, Ingrid
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    A Bioanalytical Method for Quantification of Thioredoxins in Bacillus anthracis by Digestion with Immobilized Pepsin and LC-MS/MS and On-line LC/LC-MS/MS2016In: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 79, no 7-8, p. 383-393Article in journal (Refereed)
    Abstract [en]

    We describe a method for the quantification of proteins in a biological matrix through digestion with pepsin. Pepsin is a gastric protease that efficiently cleaves proteins in an acidic environment. In this study, it has been used to generate peptides used for the quantification of physiologically relevant thioredoxin proteins in a lysate of Bacillus anthracis-the causative agent of anthrax. Carefully selected signature peptides for proteins that were digested with pepsin were immobilized on agarose gel. Filtered samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and by two-dimensional liquid chromatography tandem mass spectrometry (LC/LC-MS/MS) when additional selectivity was needed. Some important incubation parameters were adjusted to get the highest possible peptide yield. Escherichia coli was used as a surrogate matrix for the method development. The method was validated at a low nM range for selectivity, accuracy and precision. Validation showed that signature peptides were selective for the proteins, and that the method accuracy varied between 89 and 115 % with a precision of less than 12 %. The results from using pepsin in analyzing samples from Bacillus anthracis were similar to those previously obtained using western blot, and they validate pepsin as a suitable protease to generate signature peptides in a complex biological matrix as an alternative to trypsin.

  • 20.
    Sjöberg, Britt-Marie
    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.
    Radical Enzymes2001In: Encyclopedia of Life SciencesArticle, review/survey (Other (popular science, discussion, etc.))
  • 21.
    Sjöberg, Britt-Marie
    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.
    Thiols in redox mechanism of ribonucleotide reductase.2002In: Methods Enzymol, ISSN 0076-6879, Vol. 348, p. 1-21Article in journal (Other academic)
  • 22.
    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.

  • 23.
    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.

  • 24. 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.))
  • 25.
    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.

1 - 25 of 25
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