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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.
    Andersson, Martin E
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rinaldo-Matthis, Agnes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Blodig, Wolfgang
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liang, Yuhe
    Persson, Bert Ove
    Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Su, Xiao-Dong
    Nordlund, Pär
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2.2004In: Biochemistry, ISSN 0006-2960, Vol. 43, no 24, p. 7966-72Article in journal (Refereed)
    Abstract [en]

    The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.

  • 3. 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.

  • 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.
    Calmfors, Lars
    et al.
    Stockholm University, Faculty of Social Sciences, Institute for International Economic Studies.
    Heldin, Carl-Henrik
    Kragic Jansfelt, Danica
    Larsson, Mats
    Lidén, Kerstin
    Stockholm University, Faculty of Humanities, Department of Archaeology and Classical Studies, Archaeological Research Laboratory.
    Lidin, Sven
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öquist, Gunnar
    Dåliga jobbvillkor gör att Sverige tappar elitforskare2014In: Dagens nyheter, ISSN 1101-2447Article in journal (Other (popular science, discussion, etc.))
  • 6.
    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)
  • 7.
    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.

  • 8.
    Crona, Mikael
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Furrer, Ernst
    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.
    Edgell, David R.
    Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario,.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Subunit and small-molecule interaction of ribonucleotide reductases via surface plasmon resonance biosensor analyses2010In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 23, no 8, p. 633-641Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) synthesizes deoxyribonucleotides for DNA replication and repair and is controlled by sophisticated allosteric regulation involving differential affinity of nucleotides for regulatory sites. We have developed a robust and sensitive method for coupling biotinylated RNRs to surface plasmon resonance streptavidin biosensor chips via a 30.5 Å linker. In comprehensive studies on three RNRs effector nucleotides strengthened holoenzyme interactions, whereas substrate had no effect on subunit interactions. The RNRs differed in their response to the negative allosteric effector dATP that binds to an ATP-cone domain. A tight RNR complex was formed in Escherichia coli class Ia RNR with a functional ATP cone. No strengthening of subunit interactions was observed in the class Ib RNR from the human pathogen Bacillus anthracis that lacks the ATP cone. A moderate strengthening was seen in the atypical Aeromonas hydrophila phage 1 class Ia RNR that has a split catalytic subunit and a non-functional ATP cone with remnant dATP-mediated regulatory features. We also successfully immobilized a functional catalytic NrdA subunit of the E.coli enzyme, facilitating study of nucleotide interactions. Our surface plasmon resonance methodology has the potential to provide biological insight into nucleotide-mediated regulation of any RNR, and can be used for high-throughput screening of potential RNR inhibitors

  • 9. Crona, Mikael
    et al.
    Hofer, Anders
    Astorga-Wells, Juan
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tholander, Fredrik
    Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0134293Article in journal (Refereed)
    Abstract [en]

    The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related gamma-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb) 2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.

  • 10.
    Crona, Mikael
    et al.
    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.
    Moffatt, Connor
    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
    Edgell, David R.
    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
    Friedrich, Nancy C.
    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
    Hofer, Anders
    Department of Medical Biochemistry and Biophysics, Umeå University.
    Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 4, p. 1381-1389Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)2 large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

  • 11.
    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)
  • 12.
    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.

  • 13.
    Ekberg, Monika
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Birgander, Pernilla
    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.
    In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase.2003In: J Bacteriol, ISSN 0021-9193, Vol. 185, no 4, p. 1167-73Article in journal (Other academic)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.

  • 14. 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.

  • 15. Hofer, Anders
    et al.
    Crona, Mikael
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Logan, Derek T
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    DNA building blocks: keeping control of manufacture.2012In: Critical reviews in biochemistry and molecular biology, ISSN 1040-9238, E-ISSN 1549-7798, Vol. 47, no 1, p. 50-63Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α(6)β(2) complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli. Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

  • 16.
    Högbom, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huque, Yasmin
    Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Nordlund, Pär
    Department of Molecular Biology and Functional Genomics.
    Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes.2002In: Biochemistry, ISSN 0006-2960, Vol. 41, no 4, p. 1381-9Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.

  • 17. Johansson, Renzo
    et al.
    Jonna, Venkateswara Rao
    Kumar, Rohit
    Nayeri, Niloofar
    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.
    Hofer, Anders
    Logan, Derek T.
    Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 6, p. 906-917Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.

  • 18. Jonna, Venkateswara Rao
    et al.
    Crona, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rofougaran, Reza
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Samuel
    Brännström, Kristoffer
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hofer, Anders
    Diversity in Overall Activity Regulation of Ribonucleotide Reductase2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 28, p. 17339-17348Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of alpha and beta subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive alpha(6) and alpha(4)beta(4) complexes, respectively. Here we show that the Pseudomonas aeruginosa class IRNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each alpha polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an alpha(4) complex, which can interact with beta(2) to form a non-productive alpha(4)beta(2) complex. Other allosteric effectors induce a mixture of alpha(2) and alpha(4) forms, with the former being able to interact with beta(2) to form active alpha(2)beta(2) complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives.

  • 19.
    Kasrayan, Alex
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Larsson-Birgander, Pernilla
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pappalardo, Lucia
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Regnström, Karin
    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.
    Slaby, Agneta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gordon, Euan
    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.
    Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 30, p. 31050-7Article in journal (Other academic)
    Abstract [en]

    Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.

  • 20. 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)
  • 21. Kolberg, Matthias
    et al.
    Bleifuss, Günter
    Sjöberg, Britt-Marie
    Stockholm University.
    Gräslund, Astrid
    Stockholm University.
    Lubitz, Wolfgang
    Lendzian, Friedhelm
    Lassmann, Günter
    Generation and Electron Paramagnetic Resonance Spin Trapping Detection of Thinyl Radicals in Model Proteins and in the R1 Subunit of Escherichia coli2002In: Archives of Biochemistry & Biophysics, Vol. 397, p. 57-68Article in journal (Refereed)
  • 22. Kolberg, Matthias
    et al.
    Logan, Derek
    Bleifuss, Günther
    Pötsch, Stephan
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lubitz, Wolfgang
    Lassmann, Günter
    Lendzian, Friedhelm
    A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies.2005In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 12, p. 11233-46Article in journal (Other academic)
    Abstract [en]

    The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.

  • 23. Kolberg, Matthis
    et al.
    Bleifuss, Günter
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Lubitz, Wolfgang
    Lendzian, Friedhelm
    Lassmann, Günter
    Protein thiyl directly observed by EPR spectroscopy2002In: Arcives of Biochemistry & Biophysics, Vol. 403, p. 141-144Article in journal (Refereed)
  • 24.
    Larsson Birgander, Pernilla
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Bug, Stefanie
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gordon, Euan
    Dahlroth, Sue-Li
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kasrayan, Alex
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Westman, MariAnn
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Euan, Gordon
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Nucleotide-dependent formation of catalytically competent dimers from engineered monomeric ribonucleotide reductase protein R12005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 15, p. 14997-15003Article in journal (Refereed)
    Abstract [en]

    Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (α2) and R2 (β2), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.

  • 25.
    Larsson-Birgander, Pernilla
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Kasrayan, Alex
    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.
    Mutant R1 proteins from Escherichia coli class Ia ribonucleotide reductase with altered responses to dATP inhibition.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 15, p. 14496-501Article in journal (Other academic)
    Abstract [en]

    Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.

  • 26.
    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.

  • 27. Logan, Derek
    et al.
    Mulliez, Etienne
    Larsson, Karl-Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bodevin, Sabrina
    Department of Molecular Biology and Functional Genomics.
    Atta, Mohamed
    Garnaud, Pierre E
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Fontecave, Marc
    A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase.2003In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 100, no 7, p. 3826-31Article in journal (Other academic)
    Abstract [en]

    A Zn(Cys)(4) center has been found in the C-terminal region of the crystal structure of the anaerobic class III ribonucleotide reductase (RNR) from bacteriophage T4. The metal center is structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin. Mutant enzymes of the homologous RNR from Escherichia coli, in which the coordinating cysteines, conserved in almost all known class III RNR sequences, have been mutated into alanines, are shown to be inactive as the result of their inability to generate the catalytically essential glycyl radical. The possible roles of the metal center are discussed in relationship to the currently proposed reaction mechanism for generation of the glycyl radical in class III RNRs.

  • 28.
    Lundin, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gribaldo, Simonetta
    Unite Biologie Moléculaire du Gène chez les Extremophiles (BMGE), Departement de Microbiologie, Institut Pasteur, Paris, France.
    Torrents, Eduard
    Institute for Bioengineering of Catalonia (IBEC), Scientific Park of Barcelona, Barcelona, Spain.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ribonucleotide reduction: horizontal transfer of a required function spans all three domains2010In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 10, no 383Article in journal (Other academic)
    Abstract [en]

    Background Ribonucleotide reduction is the only de novo pathway for synthesis ofdeoxyribonucleotides, the building blocks of DNA. The reaction is catalysed byribonucleotide reductases (RNRs), an ancient enzyme family comprised of threeclasses. Each class has distinct operational constraints, and are broadly distributedacross organisms from all three domains, though few class I RNRs have beenidentified in archaeal genomes, and classes II and III likewise appear rare acrosseukaryotes. In this study, we examine whether this distribution is best explained bypresence of all three classes in the Last Universal Common Ancestor (LUCA), or byhorizontal gene transfer (HGT) of RNR genes. We also examine to what extentenvironmental factors may have impacted the distribution of RNR classes.

    Results Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possesseda class I RNR, but that the eukaryotic class I enzymes are not directly descended fromclass I RNRs in archaea. Instead, our results indicate that archaeal class I RNR geneshave been independently transferred from bacteria on two occasions. While LECApossessed a class I RNR, our trees indicate that this is ultimately bacterial in origin.We also find convincing evidence that eukaryotic class I RNR has been transferred tothe bacteroidetes, providing a stunning example of HGT from eukaryotes back tobacteria. Based on our phylogenies and available genetic and genomic evidence, classII and III RNRs in eukaryotes also appear to have been transferred from bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of archaeaand eukaryotes appears, through a process of elimination, to have been a dimeric classII RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT.

    Conclusions Horizontal gene transfer has clearly played an important role in the evolution of theRNR repertoire of organisms from all three domains of life. Our results clearly showthat class I RNRs have spread to archaea and eukaryotes via transfers from thebacterial domain, indicating that class I likely evolved in the bacteria. We find noclear evolutionary trace placing either class II or III RNRs in the LUCA, despite thefact that ribonucleotide reduction is an essential cellular reaction and was pivotal tothe transition from RNA to DNA genomes. Instead, a general pattern emerges whereenvironmental and enzyme operational constraints, especially the presence or absenceof oxygen, coupled with horizontal transmission are major determinants of the RNR repertoire of genomes.

  • 29.
    Lundin, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Poole, Anthony M.
    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.
    Use of Structural Phylogenetic Networks for Classification of the Ferritin-like Superfamily2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 24, p. 20565-20575Article in journal (Refereed)
    Abstract [en]

    In the postgenomic era, bioinformatic analysis of sequence similarity is an immensely powerful tool to gain insight into evolution and protein function. Over long evolutionary distances, however, sequence-based methods fail as the similarities become too low for phylogenetic analysis. Macromolecular structure generally appears better conserved than sequence, but clear models for how structure evolves over time are lacking. The exponential growth of three-dimensional structural information may allow novel structure-based methods to drastically extend the evolutionary time scales amenable to phylogenetics and functional classification of proteins. To this end, we analyzed 80 structures from the functionally diverse ferritin-like superfamily. Using evolutionary networks, we demonstrate that structural comparisons can delineate and discover groups of proteins beyond the twilight zone where sequence similarity does not allow evolutionary analysis, suggesting that considerable and useful evolutionary signal is preserved in three-dimensional structures.

  • 30.
    Lundin, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony
    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.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The functional diversity and evolutionary relationships of ferritin-like proteinsManuscript (preprint) (Other academic)
    Abstract [en]

    BackgroundThe ferritin-like proteins are evolutionarily related, as evidenced by the topology oftheir signature metal-binding four-helix bundle. They perform diverse functions suchas iron/oxygen detoxification, iron storage, DNA protection and substrate oxidation.Though evolutionarily related, sequence similarity between families and often withinfamilies is low. To analyse the evolutionary relationships between individual familiesand their functional roles systematically, we turned to 3D structural alignment andphylogenetic methods.ResultsOur phylogenetic network recovers all characterised functional groups of ferritin-likeproteins and suggests evolutionary relationships between them. The evolutionaryhypotheses that are suggested by the phylogeny are tested against availableindependent evidence such as dimerisation geometries, qualitative comparison ofstructures and sequence based partial phylogenies. Generally our hypotheses stand upagainst the evidence, but in a few cases verification have to await further data.ConclusionsTwo large evolutionary groups of ferritin-like proteins are identified from structuralphylogeny: ferritins, bacterioferritins and Dps proteins on one hand, and substrateoxidising proteins, bacterial multicomponent monooxygenases, fatty acid desaturasesand class I ribonucleotide reductase radical generating subunits, on the other. Themethod we present provides a robust way of evolutionarily classifying a functionallydiverse group of distantly related proteins, as well as examining the possible functionsof poorly-characterised proteins.

  • 31. 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.

  • 32.
    Nord, David
    et al.
    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.
    Unconventional GIY-YIG homing endonuclease encoded in group I introns in closely related strains of the Bacillus cereus group2008In: Nucleic Acids Research, Vol. 36, no 1, p. 300-310Article in journal (Refereed)
  • 33.
    Nord, David
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Torrents, Eduard
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A functional homing endonuclease in the Bacillus anthracis nrdE group I intron2007In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 189, no 14, p. 5293-5301Article in journal (Refereed)
    Abstract [en]

    The essential Bacillus anthracis nrdE gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the nrdE pre-mRNA is spliced and that the homing endonuclease cleaves an intronless nrdE gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3' extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly, nrdE genes from several other Bacillaceae were also susceptible to cleavage, with those of Bacillus cereus, Staphylococcus epidermidis (nrdE1), B. anthracis, and Bacillus thuringiensis serovar konkukian being better substrates than those of Bacillus subtilis, Bacillus lichenformis, and S. epidermidis (nrdE2). On the other hand, nrdE genes from Lactococcus lactis, Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium ammoniagenes were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase nrdE gene is a frequent target for self-splicing IVSs. A comparison of nrdE genes from seven gram-positive low-G + C bacteria, two bacteriophages, and Nocardia farcinica showed five different insertion sites for self-splicing IVSs within the coding region of the nrdE gene.

  • 34.
    Poole, Anthony
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Logan, Derek T
    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 evolution of the ribonucleotide reductases: much ado about oxygen.2002In: J Mol Evol, ISSN 0022-2844, Vol. 55, no 2, p. 180-96Article in journal (Other academic)
    Abstract [en]

    Ribonucleotide reduction is the only known biological means for de novo production of deoxyribonucleotides, the building blocks of DNA. These are produced from ribonucleotides, the building blocks of RNA, and the direction of this reaction has been taken to support the idea that, in evolution, RNA preceded DNA as genetic material. However, an understanding of the evolutionary relationships among the three modern-day classes of ribonucleotide reductase and how the first reductase arose early in evolution is still far off. We propose that the diversification of this class of enzymes is inherently tied to microbial colonization of aerobic and anaerobic niches. The work is of broader interest, as it also sheds light on the process of adaptation to oxygenic environments consequent to the evolution of atmospheric oxygen.

  • 35.
    Poole, Anthony
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Penny, David
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Confounded cytosine! Tinkering and the evolution of DNA2001In: Nature Reviews, Vol. 2, p. 147-151Article, book review (Other academic)
  • 36. 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)
  • 37.
    Rofougaran, Reza
    et al.
    Department of Medical Biochemistry and Biophysics; Umeå University.
    Crona, Mikael
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Vodnala, Munender
    Department of Medical Biochemistry and Biophysics, Umeå University.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hofer, Anders
    Department of Medical Biochemistry and Biophysics, Umeå University.
    Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, p. 35310-35318Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (α) and R2 (β). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited α4β4 complex in the presence of dATP and an active α2β2 complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30–50 times stronger in the α4β4 complex than in the α2β2 complex, which was in equilibrium with free α2 and β2 subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form α4β4 complexes. ATP could also induce the formation of a generally inhibited α4β4 complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for α4β4 formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/dTTP and forms active and inactive α6β2 complexes

  • 38.
    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.

  • 39.
    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.

  • 40.
    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.

  • 41.
    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.

  • 42.
    Sandegren, Linus
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Nord, David
    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.
    SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages.2005In: Nucleic Acids Res, ISSN 1362-4962, Vol. 33, no 19, p. 6203-13Article in journal (Other academic)
    Abstract [en]

    T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.

  • 43.
    Sandegren, Linus
    et al.
    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.
    Distribution, Sequence Homology, and Homing of Group I Introns among T-even-like Bacteriophages: Evidence for recent transfer of old introns2004In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 279, no 21, p. 22218-22227Article in journal (Refereed)
    Abstract [en]

    Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a “full set” of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.

  • 44.
    Sandegren, Linus
    et al.
    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.
    Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 21, p. 22218-27Article in journal (Other academic)
    Abstract [en]

    Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.

  • 45.
    Sandegren, Linus
    et al.
    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.
    Self-splicing of the bacteriophage T4 group I introns requires efficient translation of the pre-mRNA in vivo and correlates with the growth state of the infected bacterium.2007In: J Bacteriol, ISSN 0021-9193, Vol. 189, no 3, p. 980-90Article in journal (Other academic)
    Abstract [en]

    Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns.

  • 46.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    A Never-Eding Story2010In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 329, no 5998, p. 1475-1476Article, review/survey (Other academic)
  • 47.
    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.))
  • 48.
    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)
  • 49.
    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.

  • 50.
    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.

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