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Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter
(2022).
Diversity in myoglobin function.
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Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter
(2022).
Redox protection of pathogens by low molecular weight thiols.
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Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter
(2020).
The Crystal Structures of Bacillithiol Disulfide Reductase YpdA Reveal Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductases.
-
-
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Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter
(2019).
Enzyme Activation by a Flavoprotein Redox Network.
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Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; van Beek, Wouter & Hersleth, Hans-Petter
(2019).
New structural insight into the well known peptide flip observed in flavodoxins.
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Hammerstad, Marta; Zaltariov, Mirela F.; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W. & Cazacu, Maria
[Vis alle 14 forfattere av denne artikkelen]
(2019).
New thiosemicarbazone derivatives and their copper(II) complexes as potential inhibitors against mammalian ribonucleotide reductase.
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Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter
(2019).
Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
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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.
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Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter
(2019).
Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork
.
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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.
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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.
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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.
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Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter
(2018).
Enzyme activation by a flavoprotein redox network.
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Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer
(2017).
A structural and functional investigation of ribonucleotide reductase class III.
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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.
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Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter
(2017).
Enzyme activation by a flavoprotein redox network
.
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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.
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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.
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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.
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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.
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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.
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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.
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Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer
(2017).
A structural and functional investigation of ribonucleotide reductase class III in Bacillus cereus.
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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.
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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.
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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.
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Zaltariov, Mirela-Fernanda; Hammerstad, Marta; Arabshahi, Homayon John; Jovanović, Katarina; Richter, Klaus W. & Cazacu, Maria
[Vis alle 13 forfattere av denne artikkelen]
(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.
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Hammerstad, Marta
(2017).
New iminodiacetate-thiosemicarbazone hybrids and their copper(II) complexes are potential mR2 RNR inhibitors with high antiproliferative activity.
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Hammerstad, Marta; Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter & Zaltariov, Mirela-Fernanda
[Vis alle 9 forfattere av denne artikkelen]
(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
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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.
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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.
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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
.
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Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I.
[Vis alle 7 forfattere av denne artikkelen]
(2016).
A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE BY CD AND MCD SPECTROSCOPY.
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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.
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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.
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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.
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Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer
(2016).
A Structural and Functional Investigation of Ribonucleotide
Reductase Class III In Bacillus Cereus.
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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.
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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.
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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.
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Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I.
[Vis alle 7 forfattere av denne artikkelen]
(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.
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Hammerstad, Marta; Hersleth, Hans-Petter; Lofstad, Marie; Johannesen, Hedda; Tomter, Ane Berg & Røhr, Åsmund Kjendseth
[Vis alle 7 forfattere av denne artikkelen]
(2016).
Structural Insight into the Function of Ribonucleotide Reductase.
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Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hammerstad, Marta; Hersleth, Hans-Petter & Solomon, Edward I.
[Vis alle 7 forfattere av denne artikkelen]
(2015).
A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy.
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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.
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Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter
(2015).
Structural and functional characterization of flavohemoglobin from Bacillus Cereus.
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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.
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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.
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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)
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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.
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Lofstad, Marie; Gudim, Ingvild; Skråmo, Silje; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Tomter, Ane Berg
[Vis alle 8 forfattere av denne artikkelen]
(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)
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Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter
(2014).
Structural and functional characterization of flavohemoglobin from Bacillus Cereus.
-
Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter
(2014).
On the way to determine the structure of a ferredoxin in B. cereus.
-
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
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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
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Hammerstad, Marta
(2014).
Structural Insight Into the Function of Ribonucleotide Reductase.
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Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer
(2014).
Nitric oxide synthase and possible redox partners in Bacillus cereus.
JBIC Journal of Biological Inorganic Chemistry.
ISSN 0949-8257.
19,
s. S265–S265.
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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.
JBIC Journal of Biological Inorganic Chemistry.
ISSN 0949-8257.
19,
s. S268–S268.
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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.
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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.
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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.
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Hammerstad, Marta; Tomter, Ane Berg; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Zoppellaro, Giorgio & Andersen, Niels Højmark
[Vis alle 12 forfattere av denne artikkelen]
(2013).
Studies of the tyrosyl radicals and metal clusters in R2 of class Ia and Ib ribonucleotide reductase.
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Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer
(2013).
Nitric oxide synthase and possible redox partners in Bacillus cereus.
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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.
-
Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Lofstad, Marie; Hammerstad, Marta; Skråmo, Silje & Andersen, Niels Højmark
[Vis alle 13 forfattere av denne artikkelen]
(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.
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Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer
(2013).
Nitric oxide synthase and possible redox partners in Bacillus cereus.
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Tomter, Ane Berg; Zoppellaro, Giorgio; Hammerstad, Marta; Barra, Anne-Laure; Sandvik, Guro Katrine & Andersen, Niels Højmark
[Vis alle 15 forfattere av denne artikkelen]
(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
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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
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Hammerstad, Marta
(2013).
Crystal Structure of the B. cereus Class Ib Ribonucleotide Reductase NrdF Subunit in Complex with the Flavoprotein NrdI.
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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.
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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.
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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
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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.
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Tomter, Ane Berg; Zoppellaro, Giorgio; Hammerstad, Marta; Barra, Anne-Laure; Sandvik, Guro Katrine & Andersen, Niels Højmark
[Vis alle 11 forfattere av denne artikkelen]
(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)
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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.
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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.
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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.
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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.
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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.
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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.
JBIC 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.
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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.
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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.
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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.
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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
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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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Gudim, Ingvild; Hersleth, Hans-Petter; Hammerstad, Marta & Sørlie, Morten
(2018).
Characterisation of flavodoxin and ferredoxin/flavodoxin reductases from Bacillus cereus and their interactions.
Reprosentralen, University of Oslo.
ISSN 1501-7710.
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Shoor, Marita; Hersleth, Hans-Petter; Gudim, Ingvild & Hammerstad, Marta
(2017).
Structural and functional characterization of the redox protein Thioredoxin reductase from Bacillus cereus.
Universitetet i Oslo.
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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.
Universitet i Oslo.
ISSN 1501-7710.
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Monka, Susanne; Andersson, K. Kristoffer; Hersleth, Hans-Petter & Hammerstad, Marta
(2015).
Structural and functional characterisation of ferredoxins in Bacillus cereus.
Universitetet i Oslo.
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Wu, Bernt; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer
(2015).
Purification and characterization of Flavohemoglobin
A flavoheme enzyme
.
Universitetet i Oslo.
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Hammerstad, Marta & Andersson, K. Kristoffer
(2010).
Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants.
Universitet i Oslo.