Marta Hammerstad

• Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). A Research-Inspired Biochemistry Laboratory Module - Combining Expression, Purification, Crystallization, Structure-Solving, and Characterization of a Flavodoxin-like Protein. Biochemistry and Molecular Biology Education.  ISSN 1470-8175.  47(3), s 318- 332 . doi: 10.1002/bmb.21218
• Shi, Chunlin; Alling, Renate; Hammerstad, Marta & Aalen, Reidunn B. (2019). Control of organ abscission and other cell separation processes by evolutionary conserved peptide signaling. Plants.  ISSN 2223-7747.  8:225(7), s 1- 15 . doi: 10.3390/plants8070225
• Gudim, Ingvild; Hammerstad, Marta; Lofstad, Marie & Hersleth, Hans-Petter (2018). Characterization of different flavodoxin reductase-flavodoxin (FNR-Fld) interactions reveals an efficient FNR-Fld redox pair and identifies a novel FNR subclass. Biochemistry.  ISSN 0006-2960.  57(37), s 5427- 5436 . doi: 10.1021/acs.biochem.8b00674
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST). Bio-protocol.  ISSN 2331-8325.  7(8), s e2223 . doi: 10.21769/BioProtoc.2223 Fulltekst i vitenarkiv.
• Zaltariov, Mirela F.; Hammerstad, Marta; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W.; Cazacu, Maria; Shova, Sergiu; Balan, Mihaela; Andersen, Niels Højmark; Radulovic, Sinisa; Reynisson, Jóhannes; Andersson, Karl Kristoffer & Arion, Vladimir B (2017). New Iminodiacetate–Thiosemicarbazone Hybrids and Their Copper(II) Complexes Are Potential Ribonucleotide Reductase R2 Inhibitors with High Antiproliferative Activity. Inorganic Chemistry.  ISSN 0020-1669.  56, s 3532- 3549 . doi: 10.1021/acs.inorgchem.6b03178 Fulltekst i vitenarkiv.
• Lofstad, Marie; Gudim, Ingvild; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2016). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus. Biochemistry.  ISSN 0006-2960.  55(36), s 4998- 5001 . doi: 10.1021/acs.biochem.6b00699
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Crystal Structure of Bacillus cereus Class Ib Ribonucleotide Reductase Di-iron NrdF in Complex with NrdI. ACS Chemical Biology.  ISSN 1554-8929.  9(2), s 526- 537 . doi: 10.1021/cb400757h Vis sammendrag

  Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (β2) subunit to initiate ribonucleotide reduction in the NrdE (α2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y•) cofactor, which can self-assemble from FeII2-NrdF and O2, generation of the MnIII2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein–protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus MnII2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the dimetal site and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF dimetal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested α2β2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved.

• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Andersen, Niels Højmark; Gräslund, Astrid; Högbom, Martin & Andersson, K. Kristoffer (2014). The Class Ib Ribonucleotide Reductase from Mycobacterium tuberculosis has Two Active R2F Subunits. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  19(6), s 893- 902 . doi: 10.1007/s00775-014-1121-x
• Skråmo, Silje; Hersleth, Hans-Petter; Hammerstad, Marta; Andersson, K. Kristoffer & Røhr, Åsmund Kjendseth (2014). Cloning, expression, purification, crystallisation and preliminary X-Ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus. Acta Crystallographica. Section F : Structural Biology and Crystallization Communications.  ISSN 1744-3091.  70(No. 6. (May 2014)), s 777- 780 . doi: 10.1107/S2053230X14008334 Vis sammendrag

  A ferredoxin/flavodoxin-NADP(H) oxidoreductase (FNR) from Bacillus cereus (Bc0385) belonging to the thioredoxin reductase-like class of FNRs, has been cloned, overexpressed, purified and crystallised. Diffraction data has been collected to 2.5 Å. Abstract [150 words] Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNR) are key enzymes involved in catalysing the electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. In Bacillus cereus there are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallised in its NADPH/NADP+ free from. Diffraction data has been collected to 2.5 Å of crystals that belong to the orthorhombic space group P21212 with unit cell parameters a = 57.2, b = 164.3, c = 95.0 Å containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.

• Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Tuning of Thioredoxin Redox Properties by Intramolecular Hydrogen Bonds. PLOS ONE.  ISSN 1932-6203.  8(7) . doi: 10.1371/journal.pone.0069411 Fulltekst i vitenarkiv. Vis sammendrag

  Thioredoxin-like proteins contain a characteristic C-x-x-C active site motif and are involved in a large number of biological processes ranging from electron transfer, cellular red-ox level maintenance, and regulation of cellular processes. The mechanism for deprotonation of the buried C-terminal active site cysteine in thioredoxin, necessary for dissociation of the mixeddisulfide intermediate that occur under thiol/disulfide mediated electron transfer, is not well understood for all thioredoxin superfamily members. Here we have characterized a 8.7 kD thioredoxin (BC3987) from Bacillus cereus that unlike the typical thioredoxin appears to use the conserved Thr8 side chain near the unusual C-P-P-C active site to increase enzymatic activity by forming a hydrogen bond to the buried cysteine. Our hypothesis is based on biochemical assays and thiolate pKa titrations where the wild type and T8A mutant are compared, phylogenetic analysis of related thioredoxins, and QM/MM calculations with the BC3987 crystal structure as a precursor for modeling of reduced active sites. We suggest that our model applies to other thioredoxin subclasses with similar active site arrangements.

• Tomter, Ane Berg; Zoppellaro, Giorgio; Andersen, Niels Højmark; Hersleth, Hans-Petter; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Sandvik, Guro Katrine; Strand, Kari Røren; Nilsson, Göran Erik; Bell, Caleb B.; Barra, Anne-Laure; Blasco, Emmanuelle; Le Pape, Laurent; Solomon, Edward I. & Andersson, K. Kristoffer (2013). Ribonucleotide reductase class I with different radical generating clusters. Coordination chemistry reviews.  ISSN 0010-8545.  257(1), s 3- 26 . doi: 10.1016/j.ccr.2012.05.021 Vis sammendrag

  Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to their corresponding deoxyribonucleotides. They are formed through a radical-induced reduction of ribonucleotides. Three classes of RNR generate the catalytically active site thiyl radical using different co-factors: a tyrosyl-radical in most cases (class I), homolytic cleavage of deoxyadenosyl-cobalamin (class II), or a glycyl-radical (class III), respectively. Class I RNR has a larger subunit R1/R1E containing the active site and a smaller subunit R2/R2F with (the thiyl-generating power from) a tyrosyl radical or an oxidized iron-manganese cluster and is reviewed herein. Class I is divided into subclasses, Ia (tyrosyl-radical and di-iron-oxygen cluster), Ib (tyrosyl-radical and di-manganese-oxygen cluster) and Ic (an iron-manganese cluster). Presented here is an overview of recent developments in the understanding of class I RNR: metal-ion cluster identities, novel 3D structures, magnetic-optical properties, and reaction mechanisms. It became clear in the last years that the primitive bacterial RNR sources can utilize different metal-ion clusters to fulfil function. Within class Ia that includes members from eukaryotes (mammalians, fish) and some viruses species, the presence of hydrogen bonding interactions from water at different distances with the tyrosyl-radical site can occur. This demonstrates a large versatility in the mechanism to form the thiyl radical.

Se alle arbeider i Cristin

• Andersen, Niels Højmark; Hammerstad, Marta; Zaltariov, Mirela F.; Rapta, Peter; Arion, Vladimir B. & Hersleth, Hans-Petter (2019). Interaction of Thiosemicarbazones with the Ribonucleotide Reductase R2 subunit Studied by Resonance Raman Spectroscopy.
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork.
• Andersen, Niels Højmark; Hammerstad, Marta; Zaltariov, Mirela F.; Arion, Vladimir B. & Hersleth, Hans-Petter (2018). Interaction of Thiosemicarbazones with Ribonucleotide Reductase studied by Resonance Raman Spectroscopy.
• Gudim, Ingvild; Hersleth, Hans-Petter; Hammerstad, Marta & Sørlie, Morten (2018). Characterisation of flavodoxin and ferredoxin/flavodoxin reductases from Bacillus cereus and their interactions. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 2000.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. NBS-nytt.  ISSN 0801-3535.  s 40- 40
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2018). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Enzyme activation by a flavoprotein redox network.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class IB ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib RNR by a flavodoxin reductase in B. cereus. NSB-nytt.  s 105- 105
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus.
• Hammerstad, Marta (2017). New iminodiacetate-thiosemicarbazone hybrids and their copper(II) complexes are potential mR2 RNR inhibitors with high antiproliferative activity.
• Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). A research-inspired biochemistry laboratory module – Combining, expression,purification, crystallisation, structure solving and characterisation of a flavdoxin-like protein.. NBS-nytt.  ISSN 0801-3535.  s 63- 63
• Hammerstad, Marta; Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Zaltariov, Mirela-Fernanda; Arion, Vladimir B; Solomon, Edward I. & Andersson, K. Kristoffer (2017). INHIBITION OF CLASS 1A RIBONUCLEOTIDE REDUCTASE, AND A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE. Vis sammendrag

  INHIBITION OF CLASS 1A RIBONUCLEOTIDE REDUCTASE, AND A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE Marta Hammerstad1*; Marie Lofstad1*; Lars H. Böttger2*; Åsmund Kjendseth Røhr3; Hans-Petter Hersleth1; Mirela F. Zaltariov4; Vladimir B. Arion4; Edward I. Solomon2 and K. Kristoffer Andersson1 1Department of Biosciences, University of Oslo, Pb.1066 Blindern, NO-0316 Oslo, Norway 2Department of Chemistry, Stanford University, Stanford, CA 94305, USA 3Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, NO-1432 Ås, Norway 4Institute of Inorganic Chemistry, Universität Wien, AT-1090 Vienna, Austria *These three authors contributed equally Presenting Author’s e-mail address: k.k.andersson@ibv.uio.no Ribonucleotide reductases (RNRs) are enzymes that convert RNA building blocks into DNA building blocks [1]. The reductive reaction by RNRs requires a cysteine thiyl radical, which, in the case of class Ia or Ib RNRs, is initiated by an FeIII2- or MnIII2-tyrosyl radical (Y•) cofactor in the R2 subunit of RNR. During enzymatic turnover, the cofactor is activated by oxygen, and generates a Y• that is transported from the smaller R2 subunit to the large catalytic subunit R1 of RNR, where DNA building blocks are formed. The small subunit of class Ia RNRs can be inhibited by several small compounds [2], through the inhibition of the active FeIII2- Y• cofactor. We have performed interaction studies and Kd measurements of a mammalian R2 protein with several newly synthesized compounds, and studied their potential inhibitory effect on the protein with EPR, showing promising results. Manganese catalase (MnCAT) enzymes [3] contain an active site that is similar in structure to the MnIII2 form of NrdF (the R2 subunit in class Ib RNR), characterized by a carboxylate-bridged MnIII-O-MnIII cofactor. However, it catalyzes a different reaction – the degradation of hydrogen peroxide to dioxygen and water. A still unresolved question is how these enzymes containing similar active sites can catalyze different reactions. A variety of spectroscopic methods have been used to try to resolve this question. Samples containing NrdF with active MnIII-O-MnIII cofactor have been prepared and studied by circular dichroism (CD) and magnetic CD (MCD) spectroscopy. The data show both similar and distinct features as compared to MnCAT. 1. A.B. Tomter; G. Zoppelaro; N.H. Andersen; H.-P. Hersleth; M. Hammerstad; Å.K. Røhr; G.K. Sandvik; G.E. Nilsson; C.B. Bell; A.L. Barra; E. Blasco; L. Le Pape; E.I. Solomon; and K.K. Andersson. Coord. Chem. Rev. (2013), 257, 3 2. A. Popovic-Bijelic A; C.R. Kowol; M.E.S. Lind; J. Luo; F. Himo; A.E. Enyedy; V.B. Arion; and A. Gräslund. (2011) J. Inorg. Biochem. 105, 1422-1431 3. T.C. Brunold; D.R. Gamelin; T.L. Stemmler; S.K. Mandal; W.H. Armstrong; J.E. Penner-Hahn; and E. I. Solomon. J. Am. Chem. Soc. (1998), 120, 8724

• Johannesen, Hedda; Cumar, Rohit; Hammerstad, Marta; Hersleth, Hans-Petter; Logan, Derek & Andersson, Karl Kristoffer (2017). Investigation of NrdD and NrdG from the two Firmicutes Bacillus cereus Lactococcus lactis.
• Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III in Bacillus cereus.
• Johannesen, Hedda; Kumar, Rohit; Hersleth, Hans-Petter; Hammerstad, Marta; Logan, Derek & Andersson, Karl Kristoffer (2017). A STRUCTURAL AND FUNCTIONAL INVESTIGATION OF RIBONUCLEOTIDE REDUCTASE CLASS III IN BACILLUS CEREUS.
• Lofstad, Marie; Andersson, K. Kristoffer; Hersleth, Hans-Petter; Hammerstad, Marta & Kjendseth, Åsmund Røhr (2017). Activation Pathways of the Class Ib Ribonucleotide Reductase in Bacillus cereus. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 1808.
• Shoor, Marita; Gudim, Ingvild; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Structural and functional characterization of redox proteins in an enzyme activating network involving the thioredoxin reductase in Bacillus cereus.
• Shoor, Marita; Hersleth, Hans-Petter; Gudim, Ingvild & Hammerstad, Marta (2017). Structural and functional characterization of the redox protein Thioredoxin reductase from Bacillus cereus.
• Zaltariov, Mirela-Fernanda; Hammerstad, Marta; Arabshahi, Homayon John; Jovanović, Katarina; Richter, Klaus W.; Cazacu, Maria; Shova, Sergiu; Balan, Mihaela; Radulović, Siniša; Andersen, Niels Højmark; Reynisson, Jóhannes; Andersson, K. Kristoffer & Arion, Vladimir B (2017). New iminodiacetate-thiosemicarbazone hybrids and their copper(II) complexes are potential mR2 RNR inhibitors with high antiproliferative activity. NBS-nytt.  ISSN 0801-3535.  s 104- 104
• Gudim, Ingvild; Lofstad, Marie; Andersson, K. Kristoffer; Hammerstad, Marta & Hersleth, Hans-Petter (2016). Probing enzyme activation networks ‐ structural and functional studies of flavoproteins in Bacillus cereus. NBS-nytt.  ISSN 0801-3535.  s 45- 45
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2016). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
• Hammerstad, Marta; Hersleth, Hans-Petter; Lofstad, Marie; Johannesen, Hedda; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2016). Structural Insight into the Function of Ribonucleotide Reductase.
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2016). Structural Insight into the Function of Ribonucleotide Reductase. NBS-nytt.  ISSN 0801-3535.  s 64- 64
• Hammerstad, Marta; Kjendseth, Åsmund Røhr; Andersen, Niels Højmark; Graslund, Astrid; Högbom, Martin & Andersson, K. Kristoffer (2016). The Class Ib Ribonucleotide Reductase from Mycobacterium Tuberculosis has Two Active R2f Subunits.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A Structural and Functional Investigation of Ribonucleotide Reductase Class III In Bacillus Cereus.
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus. NBS-nytt.  ISSN 0801-3535.  s 65- 65
• Johannesen, Hedda; Hersleth, Hans-Petter; Logan, Derek; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus- Investigating the interaction and mechanism of the NrdD and NrdG complex.
• Johannesen, Hedda; Hersleth, Hans-Petter; Logan, Derek; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A structural and functional investigation of Ribonucleotide reductase Class III in Bacillus cereus- Investigating the interaction and mechanism of the NrdD and NrdG complex.
• Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Hammerstad, Marta; Solomon, Edward I. & Andersson, K. Kristoffer (2016). A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE BY CD AND MCD SPECTROSCOPY.
• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Hammerstad, Marta; Solomon, Edward I. & Andersson, K. Kristoffer (2016). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy. NBS-nytt.  ISSN 0801-3535.  s 45- 45
• Lofstad, Marie; Gudim, Ingvild; Kjendseth, Åsmund Røhr; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2016). Activation of Class Ib Ribonucleotide Reductase by NRDI and Its Reductase Partner in Bacillus cereus.
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2015). Structural Insight into the Function of Ribonucleotide Reductase. NBS-nytt.  ISSN 0801-3535.  s 47- 47 Vis sammendrag

  Structural Insight into the Function of Ribonucleotide Reductase Marta Hammerstad*, Hans-Petter Hersleth*, Ane B. Tomter*, Åsmund K. Røhr*, and K. Kristoffer Andersson* *Department of Biosciences, University of Oslo, Oslo, Norway Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (2) subunit for ribonucleotide reduction in the NrdE (2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y•) cofactor, which can self-assemble from FeII2-NrdF and O2, generation of the MnIII2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in B. cereus through conserved interactions. In addition to the B. cereus NrdI-Fe2-NrdF structure, the MnII2-NrdF and Fe2-NrdF structures show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of a metal-coordinating carboxylate seems to be is linked to the metal type. This carboxylate conformation is likely vital for the NrdF di-metal site and the flavin. We also have new results regarding the Mycobacterium tuberculosis class Ib RNR. References Hammerstad M, et al., ACS Chem. Biol. 9 (2), 526–537 (2014) Tomter AB, et al., Coord. Chem. Rev. 257, 3-26 (2013) Hammerstad M., et al., J. Biol. Inorg. Chem. 19, 893-902 (2014) Andersson KK (Ed.) Ribonucleotide reductase, Nova Science Publishers, ISBN: 978-1-60456-199-9 (2008) Røhr AK, et al., Angew. Chem. Int. Ed. 49, 2324-2327 (2010) Boal AK, et al., Science. 329, 1526 (2010)

• Lofstad, Marie; Boettger, Lars H.; Røhr, Åsmund Kjendseth; Hammerstad, Marta; Solomon, Edward I. & Andersson, K. Kristoffer (2015). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy. NBS-nytt.  ISSN 0801-3535.  s 97- 97 Vis sammendrag

  A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy Marie Lofstad1, Lars Böttger2, Åsmund K. Røhr1, Marta Hammerstad1, Edward I. Solomon2 and K. Kristoffer Andersson1 1 Department of Biosciences, University of Oslo 2 Department of Chemistry, Stanford University Ribonucleotide reductases (RNRs) are enzymes that convert RNA building blocks into DNA building blocks. This reductive reaction requires a cysteine thiyl radical, which, in the case of class Ib RNRs, is initiated by an FeIII2- or MnIII2-tyrosyl radical (Y•) cofactor in the NrdF subunit of RNR. During enzymatic turnover the cofactor is activated by oxygen, and generates a Y• that is transported from the NrdF subunit to the NrdE subunit of RNR, where DNA building blocks are formed. Manganese catalase (MnCAT) enzymes contain a similar active site structure as compared to the MnIII2 form of NrdF, characterized by a carboxylate-bridged MnIII-O-MnIII cofactor. However, it catalyzes a different reaction – the degradation of hydrogen peroxide to dioxygen and water. A still unresolved question is how these enzymes containing similar active sites can catalyze different reactions. To try to resolve this question various spectroscopic methods, like circular dichroism (CD) and magnetic CD (MCD) spectroscopy, have been used. Samples containing NrdF with active MnIII-O-MnIII cofactor have been prepared and studied at Stanford University. The collected data is currently being analyzed and compared to spectroscopic data previously obtained from MnCAT [1]. [1] Brunold, T.C. et al. J. Am. Chem. Soc. 1998, 120, 8724-8738.

• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hammerstad, Marta; Hersleth, Hans-Petter; Solomon, Edward I. & Andersson, K. Kristoffer (2015). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy.
• Monka, Susanne; Andersson, K. Kristoffer; Hersleth, Hans-Petter & Hammerstad, Marta (2015). Structural and functional characterisation of ferredoxins in Bacillus cereus.
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). On the way to determine the structure of a ferredoxin in B. cereus. Vis sammendrag

  Ferredoxins are proteins responsible for the transfer of electrons from NADPH dependent ferredoxin reductases to different enzymes in bacteria that need electrons for activation. Up to now two genes for ferredoxins have been identified in B. cereus: the bcBC 2795 gene is assumed to code for a ferredoxin with a 2Fe-2S cluster as co-factor and BCbc 1483 for a 4Fe-4S cluster carrying ferredoxin. The 2Fe-2S ferredoxin gene was cloned into a pET-22b vector and overexpressed in E. coli. The purification procedure involved ammonium sulphate precipitation, ion exchange chromatography using Q Sepharose or DEAE columns, and size-exclusion chromatography with a Superdex 75 column. The protein detection with UV-Vis spectroscopy (A280 nm) presented a challenge due to the absence of tryptophan and tyrosine residues. Therefore, the protein concentration was determined by Bradford assays. The faint yellowish colour of the purified protein (106 amino acids, 11.4 kDa) indicated the presence of an apoprotein. The crystallization screening with JCSG+ Suite resulted in several hits, of which X-ray diffraction data was collected to 2.3 Å. Solving the structure by molecular replacement has so far been unsuccessful, possibly due to a large unit cell of 220 x 220 x 220 Å3. Further studies are going to involve the reconstitution of iron-sulfur clusters, further characterization and screening for new crystallization conditions.

• Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). Structural and functional characterization of flavohemoglobin from Bacillus Cereus.
• Wu, Bernt; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2015). Purification and characterization of Flavohemoglobin A flavoheme enzyme.
• Hammerstad, Marta (2014). Structural Insight Into the Function of Ribonucleotide Reductase.
• Hammerstad, Marta & Andersson, K. Kristoffer (2014). Structural and Functional Studies of Proteins in the Class Ib Ribonucleotide Reductase System. Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo.. 1527.
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Crystal Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex.
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Structural insight Into the Function of Ribonucleotide Reductase.
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex. Acta Crystallographica Section A: Foundations of Crystallography.  ISSN 0108-7673.  70, s C434- C434 Vis sammendrag

  Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex M. Hammerstad1, H. Hersleth1, A. Tomter1, Å. Røhr1, K. Andersson1 1University of Oslo, Department of Biosciences, Oslo, Norway Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y•) cofactor in their NrdF (beta2) subunit to initiate ribonucleotide reduction in the NrdE (beta2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y•) cofactor, which can selfassemble from FeII2-NrdF and O2, generation of the MnIII2-Y• cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein-protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus MnII2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the di-metal site, and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF di-metal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y• cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested alfa2beta2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved. [1] [1] M. Hammerstad, H.-P. Hersleth, A.B.Tomter et al, ACS Chem. Biol., 2014, DOI: 10.1021/cb400757h

• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). The Bacillus cereus class Ib ribonucleotide reductase NrdIFe2- NrdF protein complex structure favors a metaldependent conformational basis for cofactor activation.
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). The Bacillus cereus class lb ribonucleotide reductase Nrdl-Fe-2-NrdF protein complex structure favors a metal-dependent conformational basis for cofactor activation. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  19, s S268- S268
• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Andersen, Niels Højmark; Graslund, Astrid; Högbom, Martin & Andersson, K. Kristoffer (2014). The Class Ib Ribonucleotide Reductase from Mycobacterium tuberculosis has two Active R2F Subunits. NBS-nytt.  ISSN 0801-3535.  s 85- 85
• Lofstad, Marie; Gudim, Ingvild; Skråmo, Silje; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Tomter, Ane Berg; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Crystallisation of ferredoxin/flavodoxin-NADP(H) oxidoreductases, flavodoxins, ferredoxins and redox partners in Bacillus cereus. Vis sammendrag

  Crystallisation of ferredoxin/flavodoxin-NADP(H) oxidoreductases, flavodoxins, ferredoxins and redox partners in Bacillus cereus Lofstad M1, Gudim I1, Skråmo S1, Hammerstad, M1, Røhr ÅK1, Tomter AB1, Andersson KK1, Hersleth H-P1 1Depatment of Biosciences, Section for Biochemistry and Molecular Biology, University of Oslo, Oslo, Norway Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) catalyses the reversible redox reaction between ferredoxins (Fds) or flavodoxins (Flds) and NAD(P)+/NAD(P)H. In oxygenic photosynthetic organisms FNRs catalyse the reduction of NADP+ by photosynthetically reduced Fd, while in many heterotrophs, FNRs catalyse the NADPH-dependent reduction of Fd/Fld to provide subsequently reducing power to different Fd/Fld-dependent enzyme systems. In Bacillus cereus there are three annotated FNRs that can be redox partners for three Flds and two Fds. We have without tags purified and crystallised several of these redox proteins in Bacillus cereus, and some of their redox partners. A goal of the project is to understand the recognition, selectivity and flexibility of these FNRs, Flds and Fds with respect to each other and their redox partners. References Skråmo S, et al., Acta Cryst. F70, 777-780 (2014) Hammerstad M, et al., ACS Chem. Biol. 9, 526-537 (2014) Røhr ÅK, et al., Angew. Chem. Int. Ed. 49, 2324-2327 (2010)

• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2014). Nitric oxide synthase and possible redox partners in Bacillus cereus. Acta Crystallographica Section A: Foundations of Crystallography.  ISSN 0108-7673.  70, s C1657- C1657 Vis sammendrag

  Nitric oxide synthase and possible redox partners in Bacillus cereus M. Lofstad1, H. Hersleth1, Å. Røhr1, M. Hammerstad1, K. Andersson1 1University of Oslo, Department of Biosciences, Oslo, Norway Nitric oxide synthase (NOS), a BH4-dependent heme-enzyme, is the only enzyme that specifically produces NO in mammals. NO is produced by the NOS homodimer in two multistep reaction cycles involving electron transfer from a reducing domain to the heme active site. The importance of NO in mammals is due to its function in signalling, vasodilation and immune response. Some bacterial species also contain NOS-encoding genes, but these bacterial NOSs are differently organized – they contain no reducing domain – and their functions and mechanism are not fully resolved [1]. Bacterial NOSs are potential drug targets, because of their role in protection against antibiotics and oxidative stress in some pathogenic bacterial species (e.g. Bacillus anthracis) [2]. Flavodoxins (Flds) have been shown to be relevant redox partners for bacterial NOSs [3], but the specificity of the interaction between NOS and Flds remains poorly understood. We have investigated the NOS protein system in Bacillus cereus, whose genome encodes NOS and two Flds, by combining crystallographic and spectroscopic methods. So far the structures of the two Flds have been solved to 0.98 Å and 2.75 Å resolution, while NOS has been solved to 2.9 Å resolution. An important part of the study has been to investigate the effect of synchrotron X-ray radiation on the oxidation state and structure of the Flds, due to their radiation sensitive cofactor flavin mononucleotide (FMN). The high-resolution (0.98 Å), oxidized structure of one Fld indicates that X-rays induce structural changes around the FMN cofactor. Another important part of the study has been to gain further insight into the specificity and flexibility of the interactions between ferredoxin/flavodoxin-NADP+ reductases, Flds and NOS in Bacillus cereus, as well as the possible mechanism of bacterial NOSs. [1] J. Sudhamsu, B. Crane, Trends in microbiology, 2009, 17, 212-218, [2] I. Gusarov, K. Shatalin, M. Starodubtseva et al, Science, 2009, 325, 1380- 1384, [3] Z. Wang, R. Lawson, M. Buddha et al, Journal of Biological Chemistry, 2007, 282, 196- 202 http://www.eiseverywhere.com/image.php?acc=4087&id=283682 Keywords: Nitric oxide synthase, Flavodoxin, Radiation damage

• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2014). Nitric oxide synthase and possible redox partners in Bacillus cereus. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  19, s S265- S265
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). On the way to determine the structure of a ferredoxin in B. cereus.
• Skråmo, Silje; Lofstad, Marie; Monka, Susanne; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Structural studies of ferredoxin/flavodoxin-NADPH reductases, flavodoxins, ferredoxins and redox partners in B. cereus.
• Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Structural and functional characterization of flavohemoglobin from Bacillus Cereus.
• Hammerstad, Marta (2013). Crystal Structure of the B. cereus Class Ib Ribonucleotide Reductase NrdF Subunit in Complex with the Flavoprotein NrdI.
• Hammerstad, Marta (2013). The Bacillus cereus class Ib ribonucleotide reductase NrdI-Fe2-NrdF protein complex structure favors a metal-dependent conformational basis for cofactor activation.
• Hammerstad, Marta; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2013). Crystal Structure of the B. cereus Class Ib Ribonucleotide Reductase NrdF Subunit in Complex with the Flavoprotein NrdI. NBS-nytt.  ISSN 0801-3535.  1, s 49- 49
• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2013). The Bacillus cereus class Ib ribonucleotide reductase NrdI-Fe2-NrdF protein complex structure favors a metal-dependent conformational basis for cofactor activation.
• Hammerstad, Marta; Tomter, Ane Berg; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Zoppellaro, Giorgio; Andersen, Niels Højmark; Sandvik, Guro Katrine; Nilsson, Göran Erik; Barra, Anne-Laure; Högbom, Martin; Graslund, Astrid & Andersson, K. Kristoffer (2013). Studies of the tyrosyl radicals and metal clusters in R2 of class Ia and Ib ribonucleotide reductase.
• Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Lofstad, Marie; Hammerstad, Marta; Skråmo, Silje; Andersen, Niels Højmark; Van Beek, Wouter; Can, Mehmet; Zhao, Xiangbo; Pompidor, Guillaume; Magliozzo, Richard S.; Bren, Kara L & Andersson, K. Kristoffer (2013). Using in situ single-crystal UV-vis and Raman spectroscopy to study the effect of X-ray radiation damage on the crystal structures of haem and flavoproteins.
• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus.
• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus.
• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Structural and functional studies of nitric oxide synthase and flavodoxins in Bacillus cereus. NBS-nytt.  ISSN 0801-3535.  1, s 84- 84 Vis sammendrag

  Abstract P6, poster Structural and functional studies of nitric oxide synthase and flavodoxins in Bacillus cereus Marie Lofstad1, Hans-Petter Hersleth1, Åsmund Kjendseth Røhr1, Marta Hammerstad1 and K. Kristoffer Andersson1 1Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindern, Oslo, Norway Nitric oxide synthase (NOS) is the only protein that specifically produces NO in mammals. The NO is produced in two multistep reaction cycles involving electron deliveries to the heme active site, and its importance is due to its function in signaling, vasodilation and immune response. Some bacterial species also contain NOS-encoding genes; however, these bacterial NOSs are differently organized and their functions are not fully understood. In this project we have investigated the NOS protein system in Bacillus cereus by combining crystallographic and spectroscopic methods. The genes encoding NOS and two flavodoxins – thought to transfer reduction equivalents to the catalytic site in NOS – from Bacillus cereus were cloned and the proteins were expressed and purified. Protein crystals were obtained from the three proteins and X-ray data was collected at synchrotrons, the resulting data being used to solve the structures of one flavodoxin (resolution 0.98 Å) and NOS (resolution 2.9 Å). The high-resolution structure of the flavodoxin, in addition to data from single-crystal spectroscopy experiments, suggested that the flavin cofactor of the flavodoxin was one-electron reduced to its semiquinone state during data collection, inducing a peptide flip of a glycine residue in the backbone of the protein. This effect of radiation has not been observed in the structure of another flavodoxin protein of Bacillus cereus. Last update: 2012-11-23 10:51:36

• Tomter, Ane Berg; Zoppellaro, Giorgio; Hammerstad, Marta; Barra, Anne-Laure; Sandvik, Guro Katrine; Andersen, Niels Højmark; Røhr, Åsmund Kjendseth; Bergan, Jonas; Ellefsen, Stian; Nilsson, Göran Erik; Bell, Caleb B.; Schmitzberger, Florian; Nordlund, Pär; Solomon, Edward I. & Andersson, K. Kristoffer (2013). Spectroscopic and DFT studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase. Vis sammendrag

  Spectroscopic and dft studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase Ane B. Tomter1; Giorgio Zoppellaro1; Marta Hammerstad1; Niels H. Andersen1;Anne-Laure Barra2; Guro K. Sandvik1; Åsmund K. Roehr1; Jonas Bergan1; Stian Ellefsen1; Göran E. Nilsson1;Caleb B. Bell III 3; Florian Schmitzberger4; Pär Nordlund4; Edward I. Solomon4; and K. Kristoffer Andersson1* 1 Department of Molecular Biosciences, University of Oslo, NO-0316 Oslo (Norway), 2 Grenoble High Magnetic Field Laboratory, CNRS, Grenoble ,France 3Departmen of Chemistry, Stanford University, Stanford, CA ,USA 4Department of Med. Biochem. & Biophys., Karolinska Institutet, Stockholm, Sweden k.k.andersson@imbv.uio.no The Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the deoxyribonucleotides. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2. The small subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity [1]. We have studied the reduced state of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning, by CD, MCD, and VTVH-MCD spectroscopy [2]. Different tyrosyl radicals were analysed by electron paramagnetic resonance (EPR/HF-EPR) [3] and resonance Raman (rRaman) spectroscopy also of RNR from an anoxia tolerant vertebrate (crucian carp) and a virus. We have published structures of mouse R2 protein and integer spin EPR of di-nuclear Co and Fe clusters [4]. In the B. cereus R2, the rRaman fingerprints, and g-tensor values of tyrosyl radical is similar those featured by E. coli R2. In other R2s differences can be seen especially in the hyperfine A-tensor and g-values values, which indicate a different rotational conformation of the phenoxyl ring and presence of sometime hydrogen bonds to phenyl-oxygens. We can also observe a tyrosyl-radical interacting with a di-manganese cluster in B. cereus R2F with help of NrdI [5,6], similar to other class Ib R2F [6,7]. The manganase R2F [8, 9] has higher specific activity than iron form with natural reductant. It seems we can obtain tyrosyl-radical interacting magnetically with other metal ions as well like Co(II). The results will be compared to the studies of E. coli R2 (class Ia and Ib), mouse R2 [4], Epstein Barr Virus R2 [10], carp R2 [11,12] and p53R2 [11,12, 13]. References. [1] K.K. Andersson (Ed.), Ribonucleotide reductase, Nova Science Publishers, Inc. Hauppauge, NY, USA, ISBN: 978-1-60456-199-9, 2008; [2] A.B. Tomter, et al., Biochemistry 2008; 47, 11300; [3] K.K. Andersson, et al., J. Biol. Inorg. Chem. 2003, 8, 23; [4] K.R. Strand, et al., J. Biol. Chem., 2002, 277, 34229; 2004, 279, 46794; . Biochemistry 2003, 42, 12223; [5] Å.K. Røhr et al., Angew. Chem. Int. Ed.. 2010, 49, 2324; [6] J.A. Cortruvo and J. Stubbe, Biochemistry 2010, 49, 1297; [7] Cox et al., J. Am. Chem. Soc. 2010, 132, 11197; [8] A.B. Tomter, et al. PLoS ONE 2012, 7, e33436; [9] Crona et al, J. Biol. Chem. 2011, 286, 33053; [10] A.B. Tomter, et al., PloS ONE 2011, 6, e25022; [11] A.B. Tomter, et al. Coordin. Chem. Rev. 2013, 257, 3-26 , doi: 10.1016/j.ccr.2012.05.021; [12] G.K. Sandvik, et al. PLoS ONE. 2012, 8, e42784; [13] Wei et al., Biochemistry 2006; 45; 14043

• Hammerstad, Marta; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Högbom, Martin; Graslund, Astrid & Andersson, K. Kristoffer (2012). Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis. Vis sammendrag

  Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis Hammerstad, M. a; Hersleth, H.-P. a; Røhr, Å. K. a; Högbom, M. b; Gräslund, A. b and Andersson, K. K a a Department of Molecular Biosciences, University of Oslo, Postboks 1041, Blindern, 0316 Oslo, Norway, marta.hammerstad@imbv.uio.no b Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius Väg 16C, SE-106 91, Stockholm, Sweden Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class I RNRs (further subdivided into class Ia, Ib and Ic) require a dinuclear metal cluster for employing a radical chemistry catalytic mechanism [1]. M. tuberculosis and B. cereus (or B. anthracis) both contain the class Ib RNR, and might require either a di-iron or di-manganese metallocofactor in their radical-generating subunits, R2F2 and R2F, respectively [2,3]. The RNR systems in both organisms include a variety of interacting proteins, such as a thioredoxin-like protein and a flavoprotein [4,5]. Biochemical, spectroscopic and structural studies of some of these proteins will be presented. Also, EPR studies indicate that a second, alternate subunit, R2F1, in M. tuberculosis might serve as an additional radical-generating subunit in its class Ib RNR. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science Publishers, Inc. Hauppauge, N.Y. USA , ISBN: 978-1-60456-199-9 [2] A.B. Tomter, et al., Plos One. 2012, 7, e33436. [3] M. Crona, et al., J. Biol. Chem. 2011, 286, 33053 [4] A.B. Tomter, et al., Coordin. Chem. Rev. 2012, Submitted. [5] Å.K. Røhr, et al., Angew. Chem. Int. Ed. 2010, 49, 2324

• Hammerstad, Marta; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Högbom, Martin; Graslund, Astrid & Andersson, K. Kristoffer (2012). Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis. Vis sammendrag

  Studies of the class Ib ribonucleotide reductase system in Bacillus cereus and Mycobacterium tuberculosis M. Hammerstad1, H.-P. Hersleth1, Å.K. Røhr1, M. Högbom2, A. Gräslund2, K.K. Andersson1 1Department of Molecular Biosciences, University of Oslo, Oslo, Norway 2Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class I RNRs (further subdivided into class Ia, Ib and Ic) require a dinuclear metal cluster for employing a radical chemistry catalytic mechanism [1]. M. tuberculosis and B. cereus (or B. anthracis) both contain the class Ib RNR, and might requires either a di-iron or di-manganese metallocofactor in their radical-generating subunits, R2F2 and R2F, respectively [2,3]. The RNR systems in both organisms include a variety of interacting proteins, such as a thioredoxin-like protein and a flavoprotein [4]. Biochemical, spectroscopic and structural studies of some of these proteins will be presented here. Also, EPR studies indicate that a second, alternate subunit, R2F1, in M. tuberculosis might serve as an additional radical-generating subunit in its class Ib RNR. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [2] A.B. Tomter, et al. Plos One. 2012, 7,3,e33436. [3] M. Crona, et al. J. Biol. Chem. 2011, 286,38. [4] A.B. Tomter, et al. CCR. 2012, Submitted.

• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2012). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987.
• Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). Structural and Functional Studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus. Vis sammendrag

  Structural and Functional Studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus Marie Lofstad, Marta. Hammerstad, Inger .K. Olsbu, Åsmund .K. Røhr, Hand-Petter.-P. Hersleth and K. Kristoffer.K. Andersson Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindern, Oslo, Norway Marie Lofstad <marie.lofstad@imbv.uio.no> Nitric Oxide Synthase (NOS) is the enzyme that produces nitric oxide in the body from arginine and oxygen. In mammals three different NOS isoforms with different functions exist: endothelial eNOS, neuronal nNOS and inducible iNOS. The mammalian NOS consists of one oxygenase domain with heme/biopterin (NOSoxy), andone reductase domain (with FMN, FAD and NADPH) and can have a calmodulin domain . The reductase domain latter transports electrons to the heme. NOSoxy. Bacterial NOSs, on the other hand, consist only of an oxygenase domain, and separate proteins deliver the electrons to this domain. The possible functions of bacterial NOSs are not fully understood. We have studied a NOS-like protein from Bacillus cereus, BC5444, which is of interest since it is a close relative to the pathogen Bacillus anthracis. Like other bacterial NOSs, it is lacking a reductase domain; however, several possible reductase partners exist in the genome. Flavodoxines are most likely candidates, due to their capacity to deliver electrons through their co-factor flavin mononucleotide (FMN). Two such flavodoxines, BC1376 and BC3541, have been further investigated. How electrons are transferred by these two flavodoxines from a flavodoxin reductase to NOS, is currently not known. Another intriguing question, concerns the specificity of the flavodoxines – is one specific flavodoxin a more efficient electron donor for NOS than the other, or are they equally good? Different biochemical and structural techniques is are going to be applied in order to try to answer these questions. So far BC5444, BC3541 and BC1376 have been cloned, expressed, purified and crystallized, and the structure of the flavodoxin BC1365 has been solved at high resolution of 1Å using X-ray crystallography. Crystals of the flavodoxin BC3541 and the NOS BC5444 have diffracted to approximately 3Å. Supported by the national Ph.D. school, Biostruct.

• Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). Structural and spectroscopic characterization of the Nitric Oxide Synthase protein system in Bacillus cereus.
• Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2012). Structural studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus.
• Skråmo, Silje; Hersleth, Hans-Petter; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Tomter, Ane Berg & Andersson, K. Kristoffer (2012). Structural and spectroscopic studies of electron transport from FldR to the Ribonucleotide reductase system in B. cereus.
• Skråmo, Silje; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2012). Studies of Electron Transfer from Flavodoxin Reductase to NrdI in Bacillus cereus.
• Tomter, Ane Berg; Zoppellaro, Giorgio; Hammerstad, Marta; Barra, Anne-Laure; Sandvik, Guro Katrine; Andersen, Niels Højmark; Røhr, Åsmund Kjendseth; Nilsson, Göran Erik; Nordlund, Pär; Solomon, Edward I. & Andersson, K. Kristoffer (2012). Spectroscopic and dft studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase. Vis sammendrag

  Spectroscopic and dft studies of the tyrosyl radicals in R2F/R2/p53R2 subunits of ribonucleotide reductase Ane B. Tomter; Giorgio Zoppellaro; Marta Hammerstad, Anne-Laure Barra; Guro K. Sandvik; Niels H. Andersen; Åsmund K. Roehr; Jonas Bergan; Stian Ellefsen; Göran E. Nilsson; Caleb B. Bell III; Florian Schmitzberger; Pär Nordlund; Edward I. Solomon; and K. Kristoffer Andersson Department of Molecular Biosciences, University of Oslo, NO-0316 Oslo (Norway), Grenoble High Magnetic Field Laboratory, CNRS, Grenoble ,France Departmen of Chemistry, Stanford University, Stanford, CA ,USA Department of Med. Biochem. and Biophys., Karolinska Institutet, Stockholm, Sweden The Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to the deoxyribonucleotides. Class I RNR is oxygen dependent and consists of two non-identical homodimeric subunits, named R1 and R2. The small subunit R2 carries a stable tyrosyl radical which is necessary for enzymatic activity [1]. We have studied the reduced state of the class Ib RNR R2F enzyme from Bacillus cereus, an opportunistic pathogen causing food poisoning, by CD, MCD, and VTVH-MCD spectroscopy [2]. Different tyrosyl radicals were analysed by electron paramagnetic resonance (EPR/HF-EPR) [3] and resonance Raman (rRaman) spectroscopy also of RNR from an anoxia tolerant vertebrate (crucian carp) and a virus. We have published structures of mouse R2 protein and integer spin EPR of di-nuclear Co and Fe clusters [4]. In the B. cereus R2, the rRaman fingerprints, and g-tensor values of tyrosyl radical is similar those featured by E. coli R2. In other R2s differences can be seen especially in the hyperfine A-tensor and g-values values, which indicate a different rotational conformation of the phenoxyl ring and presence of sometime hydrogen bonds to phenyl-oxygens. We can also observe a tyrosyl-radical interacting with a di-manganese cluster in B. cereus R2F with help of NrdI [5,6], similar to other class Ib R2F [6,7]. The manganase R2F [8, 9] has higher specific activity than iron form with natural reductant. It seems we can obtain tyrosyl-radical interacting magnetically with other metal ions. The results will be compared to the studies of E. coli R2 (class Ia and Ib), mouse R2 [4], Epstein Barr Virus R2 [10], carp R2 [11,12] and p53R2 [11,12]. References. [1] K.K. Andersson (Ed.), Ribonucleotide reductase, Nova Science Publishers, Inc. Hauppauge, NY, USA, ISBN: 978-1-60456-199-9, 2008; [2] A.B. Tomter, et al., Biochemistry 2008; 47, 11300; [3] K.K. Andersson, et al., J. Biol. Inorg. Chem. 2003, 8, 23; [4] K.R. Strand, et al., J. Biol. Chem., 2002, 277, 34229; 2004, 279, 46794; [5] Å.K. Røhr et al., Angew. Chem. Int. Ed.. 2010, 49, 2324; [6] J.A. Cortruvo, J. Stubbe, Biochemistry 2010, 49, 1297; [7] Cox et al., J. Am. Chem. Soc. 2010, 132, 11197; [8] A.B. Tomter, et al. PLoS ONE 2012, 7(3), e33436; [9] Crona et al, J. Biol. Chem. 2011, 286, 33053; [10] A.B. Tomter, et al., PloS ONE 2011, 6 (9), e25022; [11] A.B. Tomter, et al. Coordin. Chem. Rev. 2012, (in press); [12] G.K. Sandvik, et al. PLoS ONE. 2012, (in press)

• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987. Vis sammendrag

  Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 M. Hammerstad1; Å. K. Røhr1; H. Hersleth1; A. B. Tomter1; K. K. Andersson1 1. University of Oslo, Oslo, Norway. Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication [1]. RNR was the first enzyme discovered to use thioredoxins (Trxs), small ubiquitous proteins containing a redox-active cysteine dithiol/cysteine disulfide. The oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate [2]. All Trx-like proteins share an overall α/β/α sandwich fold, in addition to the conserved C-X-X-C motif [3]. E. coli NrdH-redoxins, also described as the reductants of NrdE of bacterial class Ib RNR [4], have a different active site environment compared to the typical Trx [5]. A protein homologous to the NrdH-redoxins, BC3987, has been located in the B. cereus genome, showing higher activity as an electron donor for the Mn-cofactor form than the Fe-cofactor form of the class Ib RNR [6]. Sequence similarity between these two Trx-like proteins suggests a common catalytic mechanism, involving conserved Thr residues adjacent to the active site. These threonines are believed to influence the protonation state of the C-terminal Cys of the C-X-X-C motif [7]. The function and structure of BC3987 has been characterized. The crystal structures of mutant proteins have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values were performed, enhancing our understanding of the unusual catalytic mechanism regarding these Trx-like enzymes. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [2] D. Ritz, J. Beckwith, Annu. Rev. Microbiol . 2001, 55,21. [3] J.L. Martin, Nature. 1995. 3,245. [4] A. Jordan, F. Aslund, E. Pontis, P. Reichard, A. Holmgren, J. Biol. Chem. 1997, 272,18044. [5] G.B. Kallis, A. Holmgren, J. Biol. Chem. 1980, 255, 261. [6] A.B. Tomter, (2010) Ph.D. Thesis, University of Oslo. [7] Å.K. Røhr, (2010) Ph.D. Thesis, University of Oslo. Fig. Electron density map surrounding the BC3987 T53A mutant active site.

• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987. NBS-nytt.  ISSN 0801-3535.  s 111- 111 Vis sammendrag

  Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 M. Hammerstad1, Å.K. Røhr1, H.-P. Hersleth1, A.B. Tomter1 and K.K. Andersson1 1Department of Molecular Biosciences, University of Oslo, Norway Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms [1,2]. RNR was the first enzyme discovered to use thioredoxins (Trxs), small ubiquitous proteins containing a redox-active cysteine dithiol/cysteine disulfide, for the reduction of its active site cysteines. These thiol-disulfide oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate protein [3]. All thioredoxin-like proteins share an overall // sandwich fold, in addition to the conserved C-X-X-C motif [4]. The fundamental reaction mechanism for electron transfer from Trx to its substrate was proposed by Kallis and Holmgren in 1980 [5]. E. coli NrdH-redoxins, also described as the reductants of NrdE of bacterial class Ib RNR [6], have a different active site environment compared to the typical Trx, although possessing Trx functionality. A protein homologous to the NrdH-redoxins, BC3987, has been located in the B. cereus genome [7], also possibly functioning as an electron donor for class Ib RNR [8]. Sequence similarity between these two Trx-like proteins suggests a common catalytic mechanism, involving a conserved threonine residue adjacent to the active site. This threonine is believed to influence the protonation state of the C-terminal Cys of the C-X-X-C motif [7]. The function and structure of BC3987 has been characterized using various biochemical techniques. The crystal structures of two mutant proteins have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values and redox potential determination of active site cysteine thiol/disulfide measurements were performed, enhancing our understanding regarding the unusual catalytic mechanism regarding these Trx-like enzymes. References [1] M. Kolberg, K.R. Strand, P.Graff, K.K. Andersson, Biochim. Biophys. Acta. 2004, 1699, 1. [2] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [3] D. Ritz, J. Beckwith, Annu. Rev. Microbiol . 2001, 55,21. [4] J.L. Martin, Nature. 1995. 3,245. [5] G.B. Kallis, A. Holmgren, J. Biol. Chem. 1980, 255, 261. [6] A. Jordan, F. Aslund, E. Pontis, P. Reichard, A. Holmgren, J. Biol. Chem. 1997, 272,18044. [7] Å.K. Røhr, K.K. Andersson, J. Biol. Chem (Submitted). 2010. [8] A.B. Tomter, G. Zoppelaro, C.B. Bell III, A.-L. Barra, N.H. Andersen, E.I. Solomon, K.K. Andersson, J. Biol. Chem (to be Submitted). 2011

• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987. Journal of Biological Inorganic Chemistry.  ISSN 0949-8257.  16 . doi: 10.1007/s00775-011-0862-z Vis sammendrag

  Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 M. Hammerstad1; Å. K. Røhr1; H. Hersleth1; A. B. Tomter1; K. K. Andersson1 1. University of Oslo, Oslo, Norway. Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication [1]. RNR was the first enzyme discovered to use thioredoxins (Trxs), small ubiquitous proteins containing a redox-active cysteine dithiol/cysteine disulfide. The oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate [2]. All Trx-like proteins share an overall α/β/α sandwich fold, in addition to the conserved C-X-X-C motif [3]. E. coli NrdH-redoxins, also described as the reductants of NrdE of bacterial class Ib RNR [4], have a different active site environment compared to the typical Trx [5]. A protein homologous to the NrdH-redoxins, BC3987, has been located in the B. cereus genome, showing higher activity as an electron donor for the Mn-cofactor form than the Fe-cofactor form of the class Ib RNR [6]. Sequence similarity between these two Trx-like proteins suggests a common catalytic mechanism, involving conserved Thr residues adjacent to the active site. These threonines are believed to influence the protonation state of the C-terminal Cys of the C-X-X-C motif [7]. The function and structure of BC3987 has been characterized. The crystal structures of mutant proteins have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values were performed, enhancing our understanding of the unusual catalytic mechanism regarding these Trx-like enzymes. References [1] K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, New York. [2] D. Ritz, J. Beckwith, Annu. Rev. Microbiol . 2001, 55,21. [3] J.L. Martin, Nature. 1995. 3,245. [4] A. Jordan, F. Aslund, E. Pontis, P. Reichard, A. Holmgren, J. Biol. Chem. 1997, 272,18044. [5] G.B. Kallis, A. Holmgren, J. Biol. Chem. 1980, 255, 261. [6] A.B. Tomter, (2010) Ph.D. Thesis, University of Oslo. [7] Å.K. Røhr, (2010) Ph.D. Thesis, University of Oslo. Fig. Electron density map surrounding the BC3987 T53A mutant active site.

• Lofstad, Marie; Hammerstad, Marta; Olsbu, Inger Kirstine; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2011). Structural studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus. Vis sammendrag

  Structural studies of Nitric Oxide Synthase and Flavodoxines in Bacillus cereus Author list: Marie Lofstad, Marta Hammerstad, Inger K. Olsbu, Åsmund K. Røhr, Hans-Petter Hersleth, K. Kristoffer Andersson Nitric Oxide Synthase (NOS) produces NO in the body from arginine and oxygen. In mammals three different NOS isoforms exist: endothelial eNOS, neuronal nNOS and inducible iNOS. The mammalian NOS consists of one oxygenase domain with heme (NOSoxy) and one reductase domain (with FMN, FAD and NADPH) which transports electrons to NOSoxy. Bacterial NOS, on the other hand, consists only of an oxygenase domain, and have separate proteins that deliver the electrons to this domain. The redox partner of NOS in Bacillus cereus is not known, but the three flavodoxins in Bacillus cereus are the most probable candiates. In this project we will try to structurally characterize the Bacillus cereus NOS (bcNOS) system: try to determine the natural electron donors of bcNOS, find out how specific these donors are, and understand the electron transfer between bcNOS and its electron donors. The results from these studies are going to be compared with the knowledge about the mammalian NOS reaction. I have so far in my master project cloned bcNOS and two flavodoxins. The main purpose of the project is to solve the structure of these proteins, as well as performimg activity assays and spectroscopic studies. For the flavodoxin BC1376 some nice crystals that diffracted to better than 1.0 Å, have already been obtained, and I am currently working on solving the structure.

• Skråmo, Silje; Hersleth, Hans-Petter; Hammerstad, Marta; Røhr, Åsmund Kjendseth; Tomter, Ane Berg & Andersson, K. Kristoffer (2011). Structural and spectroscopic studies of electron transport from FldR to the RNR system in B. cereus. Vis sammendrag

  Structural and spectroscopic studies of electron transport from FldR to the RNR system in B. cereus Silje Skråmo1*, Hans-Petter Hersleth1, Marta Hammerstad1, Åsmund K. Røhr1, Ane B. Tomter and K. Kristoffer Andersson1 1 Department of Molecular Biosciences, University of Oslo, Norway * silje.skramo@imbv.uio.no The Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides. The aim of the project is to study how the flavodoxin reductases (FldR) BC4926 and BC0385 transport electrons via NrdI to the RNR system in Bacillus cereus and how each of the transports affects the activity of RNR. The project is accomplished by cloning (BC0385), proteine isolation (BC0385, BC4926), activity assay measurement (FldR and NrdI, FldR, NrdI and RNR), crystallisation (BC4926 and BC0385, as well as co-crystallisation with NrdI for both FldR) and crystal structure solving. The function of NrdI in the class Ib RNR system still remains unclear. To chart the mechanism of the NrdI it is important to have an assay where the function can be studied in detail. One of the two different FldR in B. cereus is transferring electrones from NADPH to NrdI. How the reduced NrdI in turn is working on the RNR system via NrdF is strongly dependent on the FldR. Therefore, by examining both FldR, it may be possible to suggest which one is working best in vitro and maybe also in vivo. This charting may lead to possibilities of studying the interaction between NrdI and NrdF and the synthethis of the metal cofactor in the system. Structure studies of the NrdI-FldR-complex are of great interest because there exists no similar structure at this point of time.

• Hammerstad, Marta & Andersson, K. Kristoffer (2010). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants.
• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2010). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants. Vis sammendrag

  Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants Marta Hammerstad,a Åsmund K. Røhr, a Hans-Petter Hersleth, a Ane B. Tomter a and K. Kristoffer Andersson a a Department of Molecular Biosciences, University of Oslo, Norway E-mail; martaham@imbv.uio.no Thioredoxin (Trx) is a small ubiquitous protein containing a redox-active cysteine dithiol/cysteine disulfide. These thiol-disulfide oxidoreductases all share an overall // sandwich fold, in addition to the conserved C-X-X-C motif [1]. The E. coli NrdH-redoxins, described as the reductants of NrdE of bacterial class Ib RNR [2], and C. pasteurianum Cp9-redoxins, involved in the reduction of various hydroperoxide substrates [3] both possess Trx functionality. However, an active site environment differing from traditional Trxs has been proposed for these enzymes. A protein homologous to the NrdH-redoxins and Cp9-redoxins has recently been located in the B. cereus genome, BC3987, with unknown function [4]. The crystal structures of two BC3987 mutants, T53A and D11W, have been solved using X-ray crystallography. Also, for better understanding of the active site chemistry performed by the above mentioned enzymes, determination of the active site cysteine pKa values and redox potential determinations of the mutants were performed. The resulting data revealed slight alternations compared to the native protein, contributing to more knowledge about the reaction mechanism performed by these Trx-like protein. References [1] J.L. Martin, Nature. 1995. 3,245. [2] M. Kolberg et al., Biochim. Biophys. Acta. 2004, 1699, 1. [3] C.M. Reynolds et al., Biochemistry. 2002. 41,1990. [4] Å.K. Røhr and K.K. Andersson, J. Biol. Chem (Submitted). 2010.

• Hammerstad, Marta; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Tomter, Ane Berg & Andersson, K. Kristoffer (2010). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 mutants. Vis sammendrag

  Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 mutants Marta Hammerstad, Åsmund K. Røhr, Hans-Petter Hersleth, Ane B. Tomter and K.Kristoffer Andersson Department of Molecular Biosciences, University of Oslo, Norway martaham@student.matnat.uio.no Ribonucleotide reductase (RNR) was the first enzyme discovered to use thioredoxin and glutaredoxins for the reduction of active site cysteins. Class 1b RNR uses in many cases a protein called NrdH for this purpose [1, 2]. Thiol-disulfide oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate protein [3]. Thioredoxin (Trx) is a small ubiquitous protein containing a redox-active cysteine dithiol/cysteine disulfide, belonging to the thioredoxin superfamily. Although members of the thioredoxin superfamily do not have a high level of sequence similarity, all enzymes share an overall structureof four-stranded β-sheet and three flanking α-helices, in addition to the conserved C-X-X-C motif [4]. The fundamental reaction mechanism for electron transfer from Trx to its substrate has been proposed [5]. As a result of the lowered pKa value observed for the N-terminal cysteine thiol (-SH) in the E.coli Trx C-G-P-C motif, it was suggested that this thiolate (-S-) could perform the initial nucleophilic attack on the substrate disulphide bond. In order for the second nucleophilic attack performed by the buried C-terminal cysteine to take place, deprotonation caused by a conserved aspartate residue in the vicinity of the active site has been proposed [6]. Examples of groups of proteins not encompassing this Asp26, posessing Trx functionality, are the E.coli NrdH-redoxins, described as the reductants of NrdE of bacterial class Ib RNR [7], and C. pasteurianum Cp9-redoxins, involved in the reduction of various hydroperoxide substrates [8]. A protein homologous to the NrdH-redoxins and Cp9-redoxins has been located in the B.cereus genome, which, even though showing significant amino acid sequence similarity with both of the above mentioned redoxins, has no known function. The assistance of a conserved threonine residue adjacent to the active site is believed to influence the protonation state of the C-terminal Cys in this small thioredoxin [9]. The function and structure of this enzyme, BC3987, has been characterized using various biochemical techniques. The crystal stuctures of 2 mutant proteins, in addition to the native protein, have been solved using X-ray crystallography. Also, determination of active site cysteine pKa values and redox potential determination of active site cysteine thiol/disulfide measurements were performed. References 1. M. Kolberg, K.R. Strand, P.Greff, K.K. Andersson, Biochim. Biophys. Acta. 2004, 1699, 1. 2. K.K. Andersson, ed. Ribonucleotide reductase. 2008, Nova Science, NY. 3. D.Ritz, J. Beckwith, Annu. Rev. Microbiol . 2001, 55,21. 4. J.L. Martin, Nature. 1995. 3,245. 5. G.B. Kallis, A. Holmgren, J. Biol. Chem. 1980, 255, 261. 6. H. Eklund, F.K. Gleason, A. Holmgren, Proteins-Structure Function and Genetic. 1991, 11,13 7. A. Jordan, F. Aslund, E. Pontis, P. Reichard, A. Holmgren, J. Biol. Chem. 1997, 272,18044. 8. C.M. Reynolds, J. Meyer, L.B. Poole, Biochemistry. 2002. 41,1990. 9. Å.K. Røhr, K.K. Andersson, J. Biol. Chem (to be resubmitted). 2010.

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