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• Shoor, Marita; Gudim, Ingvild; Hersleth, Hans-Petter & Hammerstad, Marta (2021). Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH-binding properties. FEBS Open Bio. 11(11), p. 3019–3031. doi: 10.1002/2211-5463.13289. Full text in Research Archive Show summary

  Low molecular weight (low Mr) thioredoxin reductases (TrxRs) are homodimeric NADPH-dependent dithiol flavoenzymes that reduce thioredoxins (Trxs) or Trx-like proteins involved in the activation networks of enzymes, such as the bacterial class Ib ribonucleotide reductase (RNR). During the last few decades, TrxR-like ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs) have been discovered and characterized in several types of bacteria, including those not encoding the canonical plant-type FNR. In Bacillus cereus, a TrxR-like FNR has been shown to reduce the flavodoxin-like protein NrdI in the activation of class Ib RNR. However, some species only encode TrxR, and lack the homologous TrxR-like FNR. Due to the structural similarity between TrxRs and TrxR-like FNRs, as well as variations in their occurrence in different microorganisms, we hypothesized that low Mr TrxR may be able to replace TrxR-like FNR in, for example, the reduction of NrdI. In this study, characterization of TrxR from B. cereus has revealed a weak FNR activity towards NrdI reduction. Additionally, the crystal structure shows that only one out of two binding sites of the B. cereus TrxR homodimer is occupied with NADPH, indicating a possible asymmetric co-substrate binding in TrxR.

• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2020). The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase. Biochemistry. ISSN 0006-2960. 59(51), p. 4793–4798. doi: 10.1021/acs.biochem.0c00745. Full text in Research Archive Show summary

  Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.

• 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), p. 5427–5436. doi: 10.1021/acs.biochem.8b00674. Full text in Research Archive Show summary

  Flavodoxins (Flds) are small, bacterial proteins that transfer electrons to various redox enzymes. Flavodoxins are reduced by ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs), but little is known of the FNR-Fld interaction. Here, we compare the interactions of two flavodoxins (Fld1–2), one flavodoxin-like protein (NrdI), and three different thioredoxin reductase (TrxR)-like FNRs (FNR1–3), all from Bacillus cereus. Steady-state kinetics shows that the FNR2-Fld2 electron transfer pair is particularly efficient, and redox potential measurements also indicate that this is the most favorable electron donor/acceptor pair. Furthermore, crystal structures of FNR1 and FNR2 show that the proteins have crystallized in different conformations, a closed and an open conformation, respectively. We suggest that a large-scale conformational rearrangement takes place during the FNR catalytic cycle to allow for the binding and reduction of the Fld and, subsequently, the re-reduction of the FNR by NADPH. Finally, inspection of the residues surrounding the FAD cofactor in the FNR active site shows that a key isoalloxazine ring-stacking residue is different in FNR1 and FNR2, which could explain the large difference in catalytic efficiency between the two FNRs. To date, all of the characterized TrxR-like FNRs have a residue with aromatic character stacking against the FAD isoalloxazine ring, and this has been thought to be a conserved feature of this class of FNRs. FNR1, however, has a valine in this position. Bioinformatic analysis shows that the TrxR-like FNRs can actually be divided into two groups, one group where the FAD-stacking residue has aromatic character and another group where it is valine.

• Gudim, Ingvild; Lofstad, Marie; Beek, Wouter Van & Hersleth, Hans-Petter (2018). High-resolution crystal structures reveal a mixture of conformers of the Gly61-Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus. Protein Science. ISSN 0961-8368. 27(8), p. 1439–1449. doi: 10.1002/pro.3436. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST). Bio-protocol. 7(8), p. e2223–e2223. doi: 10.21769/BioProtoc.2223. Full text in Research Archive Show summary

  This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus (Lofstad et al., 2016). NrdI is essential in the activation of the manganese-bound form of the class Ib ribonucleotide reductase (RNR) system. RNRs, in turn, are the only source of the de novo synthesis of deoxyribonucleotides required for DNA replication and repair in all living organisms.

• 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), p. 4998–5001. doi: 10.1021/acs.biochem.6b00699. Full text in Research Archive
• Ash, Philip A; Liu, Juan; Coutard, Nathan; Heidary, Nina; Horch, Marius & Gudim, Ingvild [Show all 10 contributors for this article] (2015). Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O2 and CO. Journal of Physical Chemistry B. ISSN 1520-6106. 119, p. 13807–13815. doi: 10.1021/acs.jpcb.5b04164. Full text in Research Archive

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• Hammerstad, Marta; Gudim, Ingvild; Andersen, Hilde Kristin; Aracena-Solem, Matias & Hersleth, Hans-Petter (2026). Metals and thiols in bacterial defense and survival. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Gudim, Ingvild; Gutiérrez-Fernández, Javier & Hersleth, Hans-Petter (2025). Structural and functional studies of enzymes involved in thiol-based redox pathways. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Gutiérrez-Fernández, Javier; Gudim, Ingvild & Hersleth, Hans-Petter (2024). Structures of bacilli­thiol and myco­thiol di­sulfide reductases of bacilli and mycobacteria. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. 80. doi: 10.1107/S2053273324099200. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Fernandez, Javier Gutierrez; Gudim, Ingvild & Hersleth, Hans-Petter (2024). Structures of bacillithiol and mycothiol disulfide reductases of Firmicutes and Actinobacteria. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Full text in Research Archive
• 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. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Enzyme Activation by a Flavoprotein Redox Network. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Beek, Wouter van & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins. Acta Crystallographica Section A: Foundations and Advances. A75, p. e123–e123. doi: 10.1107/S2053273319094336. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Enzyme activation by a flavoprotein redox network. Acta Crystallographica Section A: Foundations and Advances. A75, p. e128–e128. doi: 10.1107/S2053273319094282. Full text in Research Archive
• 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. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Beek, Wouter van & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork. Full text in Research Archive
• 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. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. NBS-nytt. ISSN 0801-3535. p. 40–40. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Enzyme activation by a flavoprotein redox network. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. p. 105–105. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; Kjendseth, Åsmund Røhr; Andersen, Niels Højmark; Beek, Wouter van & Andersson, K. Kristoffer [Show all 7 contributors for this article] (2017). Combining Protein X-ray crystallography and in situ single-crystal UV-Vis and Raman spectroscopy to grasph the true structure of haem- and flavoproteins. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Hersleth, Hans-Petter; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr; beek, Wouter Van & Giullaume, Pompidor [Show all 11 contributors for this article] (2016). Grasping the True Structure of Haem- and Flavoproteins – Combining X-RAY Crystallography and In Situ Single-Crystal UV-VIS and Raman Spectroscopy. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; beek, Wouter Van; Pompidor, Guillaume; Kjendseth, Åsmund Røhr & Andersson, K. Kristoffer [Show all 7 contributors for this article] (2016). Radiation damage of haem- and flavoproteins –combining X-ray crystallography and single-crystal UV-Vis and Raman spectroscopy. 9th International Workshop on X-ray Radiation Damage to Biological Crystalline Samples. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; beek, Wouter Van; Pompidor, Guillaume; Kjendseth, Åsmund Røhr & Andersson, K. Kristoffer [Show all 7 contributors for this article] (2016). Radiation damage of haem- and flavoproteins – combining X-ray crystallography and single-crystal UV-Vis and Raman spectroscopy. Full text in Research Archive
• 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. Full text in Research Archive
• 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. p. 45–45. Full text in Research Archive
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus. Full text in Research Archive
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structures of ferredoxin/flavodoxin-NADP(H) oxidoreductases in Bacillus cereus. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. A71, p. s205–s205. doi: 10.1107/S2053273315096916. Full text in Research Archive
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structure of a ferredoxin/flavodoxin-NADP(H) oxidoreductase in Bacillus cereus. NBS-nytt. ISSN 0801-3535. p. 96–96. Full text in Research Archive
• Gudim, Ingvild & Hersleth, Hans-Petter (2015). Structures of ferredoxin/flavodoxin-NADP(H) oxidoreductases in Bacillus cereus. Full text in Research Archive
• Gudim, Ingvild (2015). Probing enyzme activation - a beginner's view on MST as a tool for biophysical studies. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; Skråmo, Silje; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Tomter, Ane Berg [Show all 8 contributors for this article] (2014). Crystallisation of ferredoxin/flavodoxin-NADP(H) oxidoreductases, flavodoxins, ferredoxins and redox partners in Bacillus cereus. Full text in Research Archive Show summary

  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)

• 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. Full text in Research Archive
• 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. Full text in Research Archive

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