martaham

• Tu, Yanyun; Cheng, Lina; Liu, Xianfeng; Hammerstad, Marta; Shi, Chunlin & Meng, Sida [Vis alle 9 forfattere av denne artikkelen] (2025). SlIDL6–SlHSL1/2/3 ligand-receptor pairs regulate tomato pedicel abscission. Journal of Integrative Agriculture (JIA). ISSN 2095-3119. 25(1), s. 118–126. doi: 10.1016/j.jia.2025.03.018. Fulltekst i vitenarkiv Vis sammendrag

  Flower and fruit abscission reduce crop yield, so decreasing abscission is a significant agricultural issue. HAESA (HAE) and HAESA-like2 (HSL2) kinases and their ligand, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide, have been confirmed to be the core elements regulating floral organ abscission in Arabidopsis thaliana. Our earlier research revealed that SlIDL6, a homolog of IDA in tomato, functions similarly to AtIDA, regulating the abscission of tomato flower organs. Here, we further isolated three HAESA-like homologs, SlHSL1/2/3, which are involved in tomato flower abscission. SlHSL1/2/3 are highly expressed in the abscission zone (AZ). The knockout mutant lines of Slhsl1, Slhsl2, and Slhsl3 showed lower flower pedicel abscission than wild type (WT). The double mutant of Slhsl1Slhsl2, Slhsl1Slhsl3, and Slhsl2Slhsl3 further depressed abscission than each of the single mutant lines, while triple mutants Slhsl1Slhsl2Slhsl3 exhibited the lowest abscission, indicating that SlHSL1/2/3 mediated abscission is non-redundancy, at least partially. Treating tomato pedicel explants with SlIDL6 peptide significantly accelerated pedicel abscission in WT. However, it had little effect on the abscission rate of SlHSL1/2/3 knockout lines, indicating that SlHSL1/2/3 are the receptors of SlIDL6 in pedicel abscission. Ethylene action inhibitor 1-methylcyclopropene (1-MCP) can significantly depress the expression of SlHSL1/2/3. Ethylene can significantly accelerate the abscission of WT, while less abscission was found in SlHSL1/2/3 knockout lines. Our findings indicate that SlHSL1/2/3 can act as receptors for SlIDL6 to positively regulate tomato pedicel abscission, and the abscission regulated by SlHSL1/2/3 was partially dependent on ethylene.

• Hammerstad, Marta; Steinvik, Erlend & Hersleth, Hans-Petter (2025). The methyltransferase NmbA methylates the low-molecular weight thiol bacillithiol, and displays a specific structural architecture. Redox Biology. 88. doi: 10.1016/j.redox.2025.103937. Fulltekst i vitenarkiv Vis sammendrag

  Low-molecular-weight (LMW) thiols maintain the cellular redox balance and protect cells against reactive species, heavy metals, toxins, and antibiotics. Despite having similar metabolic functions, structurally distinct LMW thiols are widespread in nature, with bacillithiol (BSH) being the predominant LMW thiol in bacteria. The LMW thiol N-methyl-BSH (N-Me-BSH) has been identified in the green sulfur bacterium Chlorobaculum tepidum, revealing the presence of a putative S-adenosyl-l-methionine (SAM)-dependent methyltransferase (MT), NmbA, which could catalyze the final biosynthetic step of N-Me-BSH. In this study, we report biochemical evidence for NmbA's specific function as an MT of the N-atom of the BSH cysteine moiety. We also present the crystal structure of NmbA, confirming that NmbA is a Class I SAM-dependent MT, however, displaying a unique three-dimensional architecture that differs from those of other natural product MTs (NPMTs). The NmbA active site has a narrow molecular basket structure resulting from an unusual organization of the variable Cap domain, and our docking calculations suggests that it can specifically accommodate the BSH substrate. Our research provides a valuable overview of the phylogenetic distribution of N-Me-BSH in bacteria, alongside essential functional and structural insight into a new class of N-directed NPMTs. These findings contribute to the field of SAM-dependent MTs and may allow for targeting distinct bacterial defense mechanisms involving LMW thiols with potential environmental, biotechnological, and medical implications.

• Schroer, Lisa; Weirich, Sara; Hammerstad, Marta; Hersleth, Hans-Petter; Grønsberg, Ida Andrietta & Hagen, Lars [Vis alle 11 forfattere av denne artikkelen] (2025). Orthologues of the human protein histidine methyltransferase METTL9 display distinct substrate specificities. Journal of Biological Chemistry. ISSN 0021-9258. 301(7). doi: 10.1016/j.jbc.2025.110318. Fulltekst i vitenarkiv Vis sammendrag

  The human (Homo sapiens; Hs) methyltransferase (MTase) METTL9 is the first enzyme shown to generate 1-methylhistidine (π-methylhistidine) in proteins. METTL9 preferentially methylates an alternating histidine (HxH) motif, where “x” is a small, uncharged amino acid, and multiple substrates have been identified. Putative METTL9 orthologues are found in most eukaryotes, and we have here investigated the activity of such enzymes from several species, representing all five eukaryotic supergroups. The majority of the tested enzymes demonstrated in vitro MTase activity on the prototype HsMETTL9 substrates ARMC6 and DNAJB12. We also detected protein methylation activity of the Caenorhabditis elegans METTL9 which had previously been suggested to be a DNA MTase. However, METTL9 from the fruit fly (Drosophila melanogaster; Dm) and the picoplankton Ostreococcus tauri (Ot) displayed distinct substrate specificities, differing from each other and from that of HsMETTL9. These differences were observed when recombinant proteins and short peptides were used as METTL9 substrates. To further analyze substrate specificity, we used peptide arrays to systematically replace the “x” residue and the residues flanking the HxH motif in a substrate peptide. This revealed varying degrees of tolerance among the METTL9 orthologues (Hs > Dm > Ot) for substitutions at these positions. Our results show that the METTL9 orthologues, although requiring an HxH target site, have evolved different substrate specificities, likely due to differing biologically relevant substrates in the respective organisms. Furthermore, we solved the X-ray structure of OtMETTL9, revealing several differences from the previously published HsMETTL9 structures that may explain its distinct substrate specificity.

• Drevland, Ole Martin; Muinck, Eric de; Trosvik, Pål; Hammerstad, Marta; Kvitne, Kine Eide & Midtvedt, Karsten [Vis alle 8 forfattere av denne artikkelen] (2025). Microbiome-derived reactivation of mycophenolate explains variations in enterohepatic recirculation in kidney transplant recipients. Microbiome. 13(1). doi: 10.1186/s40168-025-02142-6. Fulltekst i vitenarkiv Vis sammendrag

  Background The pivotal role of microbes in drug metabolism is increasingly recognized, as variation in the gut microbiome composition between individuals has been shown to impact systemic drug exposure, efficacy and toxicity. Mycophenolate mofetil (MMF) is a cornerstone in immunosuppressive therapy following solid organ transplantation. However, dosing and tolerance are challenged by significant pharmacokinetic variability among patients, largely due to variable degrees of enterohepatic recirculation of mycophenolic acid (MPA), the active moiety of MMF. It is hypothesized that the variability in MPA recirculation is driven by gut microbiome-derived β-glucuronidase (β-GUS) mediated cleavage of MPA-glucuronide (MPAG) excreted in the bile. Here, we investigated the bidirectional interaction between MPA and the gut microbiome in kidney transplant recipients, using a combination of in vivo and in vitro data. Results We compared the fecal microbiomes of kidney transplant recipients (n = 21) both pre- and post-transplantation to healthy individuals (n = 15) using shotgun metagenomic sequencing. We also determined the individual microbiome-derived reactivation rate of MPAG to MPA and show a strong positive correlation between this reactivation rate and the degree of MPA enterohepatic recirculation in vivo. Through metagenomic analysis, the reactivation rate of MPA was linked to specific gut microbial species. In particular, specific β-GUS gene variants associated with Faecalibacterium prausnitzii showed a strong impact on the conversion of MPAG to MPA. Furthermore, our study confirmed a significant shift in microbial composition post-transplantation and revealed notable fluctuations in species such as F. prausnitzii and Akkermansia muciniphila across different time points after transplantation. Lastly, we provide evidence that the microbiome-derived reactivation rate of MPA is linked to specific beta-glucuronidase alleles. Conclusions We highlight for the first time that the ex vivo determined reactivation rate of MPA explains the variation of enterohepatic recirculation, emphasizing the important role of F. prausnitzii in this process. More broadly, our findings suggest that the gut microbiome significantly influences the degree of enterohepatic recirculation of MPA, providing valuable insights that could be relevant for optimizing individualized immunosuppressive drug dosing in transplant patients.

• Fernandez, Javier Gutierrez; Hersleth, Hans-Petter & Hammerstad, Marta (2024). The crystal structure of myco­thiol di­sulfide reductase (Mtr) provides mechanistic insight into the specific low-molecular-weight thiol reductase activity of Actinobacteria. Acta Crystallographica Section D: Structural Biology. 80(3), s. 181–193. doi: 10.1107/S205979832400113X. Fulltekst i vitenarkiv Vis sammendrag

  Low-molecular-weight (LMW) thiols are involved in many processes in all organisms, playing a protective role against reactive species, heavy metals, toxins and antibiotics. Actinobacteria, such as Mycobacterium tuberculosis, use the LMW thiol mycothiol (MSH) to buffer the intracellular redox environment. The NADPH-dependent FAD-containing oxidoreductase mycothiol disulfide reductase (Mtr) is known to reduce oxidized mycothiol disulfide (MSSM) to MSH, which is crucial to maintain the cellular redox balance. In this work, the first crystal structures of Mtr are presented, expanding the structural knowledge and understanding of LMW thiol reductases. The structural analyses and docking calculations provide insight into the nature of Mtrs, with regard to the binding and reduction of the MSSM substrate, in the context of related oxidoreductases. The putative binding site for MSSM suggests a similar binding to that described for the homologous glutathione reductase and its respective substrate glutathione disulfide, but with distinct structural differences shaped to fit the bulkier MSSM substrate, assigning Mtrs as uniquely functioning reduc- tases. As MSH has been acknowledged as an attractive antitubercular target, the structural findings presented in this work may contribute towards future anti- tuberculosis drug development.

• Hammerstad, Marta; Rugtveit, Anne Kristine; Dahlen, Sondov Åsmundson Braathen; Andersen, Hilde Kristin & Hersleth, Hans-Petter (2023). Functional Diversity of Homologous Oxidoreductases—Tuning of Substrate Specificity by a FAD-Stacking Residue for Iron Acquisition and Flavodoxin Reduction. Antioxidants. 12(6). doi: 10.3390/antiox12061224. Fulltekst i vitenarkiv Vis sammendrag

  Although bacterial thioredoxin reductase-like ferredoxin/flavodoxin NAD(P)+ oxidoreduc- tases (FNRs) are similar in terms of primary sequences and structures, they participate in diverse biological processes by catalyzing a range of different redox reactions. Many of the reactions are critical for the growth, survival of, and infection by pathogens, and insight into the structural basis for substrate preference, specificity, and reaction kinetics is crucial for the detailed understanding of these redox pathways. Bacillus cereus (Bc) encodes three FNR paralogs, two of which have assigned distinct biological functions in bacillithiol disulfide reduction and flavodoxin (Fld) reduction. Bc FNR2, the endogenous reductase of the Fld-like protein NrdI, belongs to a distinct phylogenetic cluster of homologous oxidoreductases containing a conserved His residue stacking the FAD cofactor. In this study, we have assigned a function to FNR1, in which the His residue is replaced by a conserved Val, in the reduction of the heme-degrading monooxygenase IsdG, ultimately facilitating the release of iron in an important iron acquisition pathway. The Bc IsdG structure was solved, and IsdG-FNR1 interactions were proposed through protein–protein docking. Mutational studies and bioinformatics analyses confirmed the importance of the conserved FAD-stacking residues on the respective reaction rates, proposing a division of FNRs into four functionally unique sequence similarity clusters likely related to the nature of this residue.

• 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), s. 3019–3031. doi: 10.1002/2211-5463.13289. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Furumizu, Chihiro; Krabberød, Anders Kristian; Hammerstad, Marta; Alling, Renate; Wildhagen, Mari & Sawa, Shinichiro [Vis alle 7 forfattere av denne artikkelen] (2021). The sequenced genomes of non-flowering land plants reveal the innovative evolutionary history of peptide signaling. The Plant Cell. ISSN 1040-4651. 33(9), s. 2915–2934. doi: 10.1093/plcell/koab173. Fulltekst i vitenarkiv Vis sammendrag

  Abstract An understanding of land plant evolution is a prerequisite for in-depth knowledge of plant biology. Here we extract and explore information hidden in the increasing number of sequenced plant genomes, from bryophytes to angiosperms, to elucidate a specific biological question—how peptide signaling evolved. To conquer land and cope with changing environmental conditions, plants have gone through transformations that must have required innovations in cell-to-cell communication. We discuss peptides mediating endogenous and exogenous changes by interaction with receptors activating intracellular molecular signaling. Signaling peptides were discovered in angiosperms and operate in tissues and organs such as flowers, seeds, vasculature, and 3D meristems that are not universally conserved across land plants. Nevertheless, orthologs of angiosperm peptides and receptors have been identified in nonangiosperms. These discoveries provoke questions regarding coevolution of ligands and their receptors, and whether de novo interactions in peptide signaling pathways may have contributed to generate novel traits in land plants. The answers to such questions will have profound implications for the understanding of the evolution of cell-to-cell communication and the wealth of diversified terrestrial plants. Under this perspective, we have generated, analyzed, and reviewed phylogenetic, genomic, structural, and functional data to elucidate the evolution of peptide signaling.

• Stepanenko, Iryna; Babak, Maria V.; Spengler, Gabriella; Hammerstad, Marta; Popovic-Bijelic, Ana & Shova, Sergiu [Vis alle 10 forfattere av denne artikkelen] (2021). Coumarin-Based Triapine Derivatives and Their Copper(II) Complexes: Synthesis, Cytotoxicity and mR2 RNR Inhibition Activity. Biomolecules. 11(862). doi: 10.3390/biom11060862. Fulltekst i vitenarkiv Vis sammendrag

  A series of thiosemicarbazone-coumarin hybrids (HL1-HL3 and H2L4) has been synthesised in 12 steps and used for the preparation of mono- and dinuclear copper(II) complexes, namely Cu(HL1)Cl2 (1), Cu(HL2)Cl2 (2), Cu(HL3)Cl2 (3) and Cu2(H2L4)Cl4 (4), isolated in hydrated or solvated forms. Both the organic hybrids and their copper(II) and dicopper(II) complexes were comprehensively characterised by analytical and spectroscopic techniques, i.e., elemental analysis, ESI mass spectrometry, 1D and 2D NMR, IR and UV–vis spectroscopies, cyclic voltammetry (CV) and spectroelectrochemistry (SEC). Re-crystallisation of 1 from methanol afforded single crystals of copper(II) complex with monoanionic ligand Cu(L1)Cl, which could be studied by single crystal X-ray diffraction (SC-XRD). The prepared copper(II) complexes and their metal-free ligands revealed antiproliferative activity against highly resistant cancer cell lines, including triple negative breast cancer cells MDA-MB-231, sensitive COLO-205 and multidrug resistant COLO-320 colorectal adenocarcinoma cell lines, as well as in healthy human lung fibroblasts MRC-5 and compared to those for triapine and doxorubicin. In addition, their ability to reduce the tyrosyl radical in mouse R2 protein of ribonucleotide reductase has been ascertained by EPR spectroscopy and the results were compared with those for triapine.

• Besleaga, Iuliana; Stepanenko, Iryna; Petrasheuskaya, Tatsiana V.; Darvasiova, Denisa; Breza, Martin & Hammerstad, Marta [Vis alle 14 forfattere av denne artikkelen] (2021). Triapine Analogues and Their Copper(II) Complexes: Synthesis, Characterization, Solution Speciation, Redox Activity, Cytotoxicity, and mR2 RNR Inhibition. Inorganic Chemistry. ISSN 0020-1669. 60, s. 11297–11319. doi: 10.1021/acs.inorgchem.1c01275. Fulltekst i vitenarkiv Vis sammendrag

  Three new thiosemicarbazones (TSCs) HL1–HL3 as triapine analogues bearing a redox-active phenolic moiety at the terminal nitrogen atom were prepared. Reactions of HL1–HL3 with CuCl2·2H2O in anoxic methanol afforded three copper(II) complexes, namely, Cu(HL1)Cl2 (1), [Cu(L2)Cl] (2′), and Cu(HL3)Cl2 (3), in good yields. Solution speciation studies revealed that the metal-free ligands are stable as HL1–HL3 at pH 7.4, while being air-sensitive in the basic pH range. In dimethyl sulfoxide they exist as a mixture of E and Z isomers. A mechanism of the E/Z isomerization with an inversion at the nitrogen atom of the Schiff base imine bond is proposed. The monocationic complexes [Cu(L1–3)]+ are the most abundant species in aqueous solutions at pH 7.4. Electrochemical and spectroelectrochemical studies of 1, 2′, and 3 confirmed their redox activity in both the cathodic and the anodic region of potentials. The one-electron reduction was identified as metal-centered by electron paramagnetic resonance spectroelectrochemistry. An electrochemical oxidation pointed out the ligand-centered oxidation, while chemical oxidations of HL1 and HL2 as well as 1 and 2′ afforded several two-electron and four-electron oxidation products, which were isolated and comprehensively characterized. Complexes 1 and 2′ showed an antiproliferative activity in Colo205 and Colo320 cancer cell lines with half-maximal inhibitory concentration values in the low micromolar concentration range, while 3 with the most closely related ligand to triapine displayed the best selectivity for cancer cells versus normal fibroblast cells (MRC-5). HL1 and 1 in the presence of 1,4-dithiothreitol are as potent inhibitors of mR2 ribonucleotide reductase as triapine.

• Hammerstad, Marta & Hersleth, Hans-Petter (2021). Overview of structurally homologous flavoprotein oxidoreductases containing the low Mr thioredoxin reductase-like fold – A functionally diverse group. Archives of Biochemistry and Biophysics. ISSN 0003-9861. 702, s. 1–19. doi: 10.1016/j.abb.2021.108826. Fulltekst i vitenarkiv Vis sammendrag

  Structural studies show that enzymes have a limited number of unique folds, although structurally related enzymes have evolved to perform a large variety of functions. In this review, we have focused on enzymes containing the low molecular weight thioredoxin reductase (low Mr TrxR) fold. This fold consists of two domains, both containing a three-layer ββα sandwich Rossmann-like fold, serving as flavin adenine dinucleotide (FAD) and, in most cases, pyridine nucleotide (NAD(P)H) binding-domains. Based on a search of the Protein Data Bank for all published structures containing the low Mr TrxR-like fold, we here present a comprehensive overview of enzymes with this structural architecture. These range from TrxR-like ferredoxin/flavodoxin NAD(P)+ oxidoreductases, through glutathione reductase, to NADH peroxidase. Some enzymes are solely composed of the low Mr TrxR-like fold, while others contain one or two additional domains. In this review, we give a detailed description of selected enzymes containing only the low Mr TrxR-like fold, however, catalyzing a diversity of chemical reactions. Our overview of this structurally similar, yet functionally distinct group of flavoprotein oxidoreductases highlights the fascinating and increasing number of studies describing the diversity among these enzymes, especially during the last decade(s).

• 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), s. 4793–4798. doi: 10.1021/acs.biochem.0c00745. Fulltekst i vitenarkiv Vis sammendrag

  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.

• 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. Fulltekst i vitenarkiv Vis sammendrag

  Many laboratory courses consist of short and seemingly unconnected individual laboratory exercises. To increase the course consistency, relevance, and student engagement, we havedevelopedaresearch-inspiredandproject-basedmodule, “From Gene to Structure and Function”. This 2.5-week full-day biochemistry and structural biology module covers protein expression, purification, structure solving, and characterization.Themoduleiscenteredaroundthe flavodoxin-likeprotein NrdI, involved in the activation of the bacterial ribonucleotide reductase enzyme system. Through an in-depth focus on one specific protein, the students will learn the basic laboratory skills needed in order to generate a broader knowledge and breadth within the field. With respect to generic skills, the studentsreporttheir findingsasascientificarticle,withtheaim to learn to present concise research results and write scientific papers. The current research-inspired project has the potential ofbeingfurtherdevelopedintoamorediscovery-drivenproject and extended to include other molecular biological techniques orbiochemical/biophysicalcharacterizations.Instudentevaluations, this research-inspired laboratory course has received very high ratings and been highly appreciated, where the students have gained research experience for more independent future work in the laboratory. © 2019 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry andMolecularBiology,47(3):318–332,2019.

• 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. 8(7), s. 1–15. doi: 10.3390/plants8070225. Fulltekst i vitenarkiv Vis sammendrag

  Plants both generate and shed organs throughout their lifetime. Cell separation is in function during opening of anthers to release pollen; floral organs are detached after pollination when they have served their purpose; unfertilized flowers are shed; fruits and seeds are abscised from the mother plant to secure the propagation of new generations. Organ abscission takes place in specialized abscission zone (AZ) cells where the middle lamella between adjacent cell files is broken down. The plant hormone ethylene has a well-documented promoting effect on abscission, but mutation in ethylene receptor genes in Arabidopsis thaliana only delays the abscission process. Microarray and RNA sequencing have identified a large number of genes differentially expressed in the AZs, especially genes encoding enzymes involved in cell wall remodelling and disassembly. Mutations in such genes rarely give a phenotype, most likely due to functional redundancy. In contrast, mutation in the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) blocks floral organ abscission in Arabidopsis. IDA encodes a small peptide that signals through the leucine-rich repeat receptor-like kinases HAESA (HAE) and HAE-LIKE2 (HSL2) to control floral organ abscission and facilitate lateral root emergence. Untimely abscission is a severe problem in many crops, and in a more applied perspective, it is of interest to investigate whether IDA-HAE/HSL2 is involved in other cell separation processes and other species. Genes encoding IDA and HSL2 orthologues have been identified in all orders of flowering plants. Angiosperms have had enormous success, with species adapted to all kinds of environments, adaptations which include variation with respect to which organs they shed. Here we review, from an evolutionary perspective, the properties of the IDA-HAE/HSL2 signaling module and the evidence for its hypothesized involvement in various cell separation processes in angiosperms.

• 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. Fulltekst i vitenarkiv Vis sammendrag

  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; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST). Bio-protocol. 7(8), s. e2223–e2223. doi: 10.21769/BioProtoc.2223. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Zaltariov, Mirela F.; Hammerstad, Marta; Arabshahi, Homayon John; Jovanovic, 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 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 Vis sammendrag

  As ribonucleotide reductase (RNR) plays a crucial role in nucleic acid metabolism, it is an important target for anticancer therapy. The thiosemicarbazone Triapine is an efficient R2-inhibitor, which has entered about 20 clinical trials. Thiosemicarbazones are supposed to exert their biological effects through effectively binding transition metal ions. In this study, six iminodiacetatethiosemicarbazones able to form transition metal complexes, as well as six dicopper(II) complexes were synthesized and fully characterized by analytical, spectroscopic techniques (IR; UV‒vis, 1 H and 13C NMR), ESI mass spectrometry and X-ray diffraction. The antiproliferative effects were examined in several human cancer and one noncancerous cell lines. Several of the compounds showed high cytotoxicity and marked selectivity for cancer cells. Based on this, and on molecular docking calculations one lead dicopper(II) complex and one thiosemicarbazone were chosen for in vitro analysis as potential R2 inhibitors. Their interaction with R2 and effect on the Fe(III)2-Y• cofactor were characterized by microscale thermophoresis, and two spectroscopic techniques, EPR and UV‒vis spectroscopy. Our findings suggest that several of the synthesized proligands and copper(II) complexes are effective antiproliferative agents in several cancer cell lines, targeting RNR, which deserve further investigation as potential anticancer drugs. The final version of this research has been published in Inorganic Chemistry. © 2017 American Chemical Society

• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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.

• 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. 70(No. 6. (May 2014) ), s. 777–780. doi: 10.1107/S2053230X14008334. Fulltekst i vitenarkiv 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.

• 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. JBIC Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 19(6), s. 893–902. doi: 10.1007/s00775-014-1121-x. Fulltekst i vitenarkiv
• Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Tuning of Thioredoxin Redox Properties by Intramolecular Hydrogen Bonds. PLOS ONE. 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 [Vis alle 15 forfattere av denne artikkelen] (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. Fulltekst i vitenarkiv 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 NVA

• Hammerstad, Marta; Gudim, Ingvild; Andersen, Hilde Kristin; Aracena-Solem, Matias & Hersleth, Hans-Petter (2026). Metals and thiols in bacterial defense and survival. Fulltekst i vitenarkiv
• Hammerstad, Marta & Hersleth, Hans-Petter (2025). Structural studies of flavodoxins from Bacillus cereus in different redox states. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2025). Structural and functional insights into bacillithiol disulfide reductase – A potential antimicrobial target. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2024). Structural and functional investigation of bacillithiol disulfide reductases. Fulltekst i vitenarkiv
• Hammerstad, Marta; Andersen, Hilde Kristin; Rugtveit, Anne Kristine; Dahlen, Sondov Åsmundson Braathen & Hersleth, Hans-Petter (2023). Functional diversity among flavin-containing oxidoreductases involved in redox protection, iron homeostasis, and ribonucleotide reduction. Fulltekst i vitenarkiv
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Fulltekst i vitenarkiv
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2023). Diversity among bacillithiol disulfide reductases – a structural and functional investigation. Fulltekst i vitenarkiv
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Fulltekst i vitenarkiv
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Fulltekst i vitenarkiv
• Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2023). A Research-inspired Laboratory Module on a Redox Protein Combining Biochemistry and Structural Biology. Fulltekst i vitenarkiv
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Structural determination of Mycobacterium tuberculosis and Rhodococcus erythropolis mycothiol disulphide reductases. Fulltekst i vitenarkiv
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Fulltekst i vitenarkiv
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function. Fulltekst i vitenarkiv
• Andersen, Hilde Kristin; Hersleth, Hans-Petter & Hammerstad, Marta (2022). Structure-function studies of ferredoxin-flavodoxin NADP+ oxidoreductase 1 from Bacillus cereus as an iron-uptake oxidoreductase. Fulltekst i vitenarkiv
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function. Fulltekst i vitenarkiv
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Enzyme Activation by a Flavoprotein Redox Network. Fulltekst i vitenarkiv
• 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, s. e123–e123. doi: 10.1107/S2053273319094336. Fulltekst i vitenarkiv
• 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, s. e128–e128. doi: 10.1107/S2053273319094282. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Enzyme activation by a flavoprotein redox network. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III in Bacillus cereus. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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

• Hammerstad, Marta (2017). New iminodiacetate-thiosemicarbazone hybrids and their copper(II) complexes are potential mR2 RNR inhibitors with high antiproliferative activity. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A Structural and Functional Investigation of Ribonucleotide Reductase Class III In Bacillus Cereus. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). On the way to determine the structure of a ferredoxin in B. cereus. Fulltekst i vitenarkiv 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.

• 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. Fulltekst i vitenarkiv 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.

• Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). Structural and functional characterization of flavohemoglobin from Bacillus Cereus. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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)

• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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; 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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

• Hammerstad, Marta; Hersleth, Hans-Petter; Tomter, Ane Berg; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer (2014). Structural insight Into the Function of Ribonucleotide Reductase. Fulltekst i vitenarkiv
• Skråmo, Silje; Lofstad, Marie; Monka, Susanne; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Andersson, K. Kristoffer [Vis alle 7 forfattere av denne artikkelen] (2014). Structural studies of ferredoxin/flavodoxin-NADPH reductases, flavodoxins, ferredoxins and redox partners in B. cereus. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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)

• Hammerstad, Marta (2014). Structural Insight Into the Function of Ribonucleotide Reductase. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). On the way to determine the structure of a ferredoxin in B. cereus. Fulltekst i vitenarkiv
• Wu, Bernt; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2014). Structural and functional characterization of flavohemoglobin from Bacillus Cereus. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Lofstad, Marie; Hersleth, Hans-Petter; Røhr, Åsmund Kjendseth; Hammerstad, Marta & Andersson, K. Kristoffer (2013). Nitric oxide synthase and possible redox partners in Bacillus cereus. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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 (2013). Crystal Structure of the B. cereus Class Ib Ribonucleotide Reductase NrdF Subunit in Complex with the Flavoprotein NrdI. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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

• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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.

• 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. Fulltekst i vitenarkiv 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.

• 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. Fulltekst i vitenarkiv 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; 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. Fulltekst i vitenarkiv 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

• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv 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. Fulltekst i vitenarkiv 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

• 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. Fulltekst i vitenarkiv 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.

• 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. Fulltekst i vitenarkiv 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.

• 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. Fulltekst i vitenarkiv 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 (2010). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants. Fulltekst i vitenarkiv 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. Fulltekst i vitenarkiv 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.

• Andersen, Hilde Kristin; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Struktur-funksjonsstudier av ferredoksin-flavodoksin NADP+ oksidoreduktase 1 fra Bacillus cereus som en jern-opptak oksidoreduktase. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Bacillus cereus har tre enzymer som er annotert som ferredoksin-flavodoksin NADP+ oksidoreduktaser (FNRer). FNR3 er vist å redusere disulfidformen av bacillitiol, FNR2 er vist å effektivt redusere flavodoksiner, men det er enda ikke funnet noen god redokspartner til FNR1. FNR1 er vist å skille seg fra FNR2 ved å være del av en fylogenetisk undergruppe av tioredoksinreduktase-aktige FNRer hvor en konservert aromatisk residu i C-terminalen, som stabiliserer FAD-kofaktoren er erstattet med valin sammenlignet med FNR2-gruppen. Staphylococcus aureus har en nær homolog til FNR1 som deler den konserverte valinresiduen, som har blitt vist å kunne fungere som en jern-opptak-oksidoreduktase (IruO). IruO kan tilgjengeliggjøre jern for bakterier ved å redusere hemoksygenasene iron-regulated surface determinant (Isd)G og IsdI som degraderer hem, eller sideroforer. B. cereus har kun en homolog til IsdG, men ikke IsdI. I dette prosjektet har det blitt utført aktivitetsstudier for å undersøke om B. cereus FNR1 kan fungere som en IruO, og redusere IsdG for å degradere hem, ved bruk av UV-vis-spektroskopi. FNR1 og IsdG har blitt uttrykt og renset. Strukturen til IsdG er blitt løst ved bruk av proteinkrystallografi. IsdG viste å ha svært lik struktur i B. cereus og S. aureus, med ferredoksinfolding og de konserverte residuene Asn7, Trp67 og His77 i bindingssetet til hem. Aktivitetsstudiene viste at FNR1 kunne redusere IsdG for å degradere hem og tilgjengeliggjøre jern. FNR1 er anslått å ha evner i samme størrelsesorden til å redusere IsdG som til å redusere flavodoksiner. Aktivitetsstudiene tyder på at hem blir degradert ved først å redusere Fe(III) til Fe(II) og at det går via Fe(II)-O2 som i kanoniske hemoksygenaser.

• Rese, Morten; Hersleth, Hans-Petter; Hammerstad, Marta & Andersen, Niels Højmark (2022). Functional diversity of myoglobin isozymes from Carassius auratus - Biophysical study of myoglobin as a peroxidase. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Myoglobin (Mb) is a heme protein expressed in oxidative muscle tissue and functions in oxygen storage and transport. Mb can also display complex redox chemistry involved in several important physiological processes including scavenging of reactive oxygen species and nitric oxide metabolism. Goldfish and the common carp are the only vertebrates known today that express two paralogs of Mb (Mb1 and Mb2) as a result of a recent whole genome duplication. Mb1 is expressed in gills, liver, and skeletal muscle tissue, whereas Mb2 is exclusively expressed in the brain. The tissue-specific expression led to work showing that Mb2 in carp fish is a more effective peroxidase. In this study, work has been done to gain detailed structural and functional insight into the differences of the goldfish Mb1 and Mb2 isozymes by biophysical characterization and 3D structure analysis. The intensively studied Mb from horse heart (hMb) has been studied together with Mb1 and Mb2 as a reference. The reaction of Mb with hydrogen peroxide (H2O2) has been of particular interest. In addition, studies on how radiation damage during diffraction data collection affects redox states of Mb have been performed. This study shows that goldfish Mb2 reacts with H2O2 significantly faster than Mb1. Structural modeling of Mb1 and Mb2 indicates that no differences around the active site are responsible for the difference in reactivity. The structural basis of this functional difference is therefore complex in nature and might be explained by altered dynamics as a consequence of differing amino acid composition towards the surface of the proteins. The ferric and ferryl redox states of Mb are damaged by X-ray radiation during diffraction data collection. The ferric state is significantly more susceptible to radiation damage compared to the ferryl state. This study shows the importance of performing a time/dose calculation on the amount of radiation a specific redox state can handle when collecting diffraction data.

• Hersleth, Hans-Petter; Hammerstad, Marta & Dahlen, Sondov Åsmundson Braathen (2021). Structure and function of FNR1 from Bacillus cereus undergoing a mutation. Universitetet i Oslo. Fulltekst i vitenarkiv
• Rugtveit, Anne Kristine; Hammerstad, Marta & Hersleth, Hans-Petter (2021). Structural and functional studies of a ferredoxin/flavodoxin NADP+-oxidoreductase mutant from Bacillus cereus. Universitetet i Oslo. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Wu, Bernt; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2015). Purification and characterization of Flavohemoglobin - A flavoheme enzyme. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Flavohemoglobin is a flavoheme enzyme that catalyzes dioxygenation of nitric oxide (NO) to nitrate NO3&amp;ndash;. NO is diatomic radical gas produced by enzymatic and non-enzymatic oxidation of reduced nitrogenous compounds. NO acts as a membrane-permeable signal molecule in mammals, in addition to being a toxin at higher concentrations and being utilized by the immune system against invading pathogens. FHb is involved in protection against the cytotoxic effects of NO (termed nitrosative stress) and is found in a range of bacterial species and some fungi. FHbs are not found in higher eukaryotes, but is employed by a range of pathogens, and is a potential therapeutic target. In this thesis we sought to investigate the FHb from Bacillus cereus by determination of the X-ray crystal structure as only a few structures of FHb from different organisms have been obtained. The protein was successfully cloned and expressed and a purification protocol has been developed, with ammonium sulfate precipitation, anion exchange chromatography and gel filtration. A high yield with high purity was obtained, but with heme and FAD at substoichiometric levels. Crystallization screening resulted in some small needles grown from precipitate, although they were non-reproducible and no crystal structure was obtained. A heme assay was performed in order to identify the heme/protein ratio and protein assays was performed to calculate protein concentration. Testing of reconstitution with heme and FAD was performed with promising results.

• Monka, Susanne; Andersson, K. Kristoffer; Hersleth, Hans-Petter & Hammerstad, Marta (2015). Structural and functional characterisation of ferredoxins in Bacillus cereus. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Ferredoxins are iron-sulfur proteins that transfer electrons in different cellular processes. In Bacillus cereus there are two genes for ferredoxins that have been identified, one present with a [2Fe-2S] cluster (BC2795) and one with a [4Fe- 4S] cluster (BC1483). These proteins can potentially receive electrons from three possible ferredoxin-NADP+-reductases, and further activate different enzymes. In this project, the starting point was to purify and structurally characterize the two ferredoxins in B. cereus and potentially their interactions with redox partners. For the [4Fe-4S] ferredoxin several attempts to overexpress this protein was unsuccessful. However, the [2Fe-2S] ferredoxin (106 amino acids, 11.4 kDa) was successfully overexpressed in Escherichia coli. This [2Fe-2S] ferredoxin was purified in a series of purification steps involving ammonium sulphate, desalting column, Q Sepharose or DEAE columns, and Superdex 75 column. The purification procedure showed to be troublesome because of possible low iron-sulfur cluster content, and because of low UV-vis detection due to the absence of tryptophanes and tyrosines in the protein. Due to the high content of apoprotein, it was also tried to establish a reconstitution procedure for BC2795. The apoform of the protein was crystallized. Nevertheless, it was not possible to solve the structure.

• Hammerstad, Marta & Andersson, K. Kristoffer (2014). Structural and Functional Studies of Proteins in the Class Ib Ribonucleotide Reductase System. Universitetet i Oslo. Fulltekst i vitenarkiv
• Hammerstad, Marta & Andersson, K. Kristoffer (2010). Biochemical and Structural Characterization of the Bacillus cereus Thioredoxin BC3987 Mutants. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Thioredoxin (Trx) is a small ubiquitous protein containing two conserved cysteines that can reversibely form a redox-active disulfide bond, belonging to the Trx superfamily. Ribonucleotide reductase (RNR) was the first enzyme discovered to use Trxs and glutaredoxins (Grxs) for the reduction of active site cysteines. Class Ib RNR uses in many cases a protein called NrdH-redoxin for this purpose. Thiol-disulfide oxidoreductases perform the fast and reversible thiol-disulfide exchange between their active site cysteines and cysteines in the substrate protein. Although most Trx-like proteins do not have a high level of sequence similarity, all enzymes share an overall // sandwich fold, in addition to the conserved C-X-X-C motif. The fundamental reaction mechanism for electron transfer from Trx to its substrate was proposed by Kallis and Holmgren in 1980. As a result of the lowered pKa value observed for the N-terminal cysteine thiol (-SH) in the Escherichia 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. Examples of groups of proteins not encompassing this Asp26, but possessing Trx functionality, are the E. coli NrdH-redoxins, described as the reductants of NrdE of bacterial class Ib RNR, and C. pasteurianum Cp9-redoxins, involved in the reduction of various hydroperoxide substrates. A protein homologous to the NrdH-redoxins and Cp9-redoxins has been located in the Bacillus cereus genome, showing significant amino acid sequence similarity with both of the above mentioned redoxins. However, its function is still unknown. A conserved threonine residue, Thr8, adjacent to the active site is believed to influence the protonation state of the C-terminal cysteine in the active site of this small Trx. Other residues might influence the protonation state of this residue as well. The function and structure of this enzyme, BC3987, has been characterized using various biochemical techniques. The crystal structures of two mutant proteins; BC3987 D11W and T53A, in addition to the native protein, have been solved using X-ray crystallography. Also, determinations of active site cysteine pKa values and redox potentials of active site cysteine thiols/disulfides were performed.

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