martaham

• Tu, Yanyun; Cheng, Lina; Liu, Xianfeng; Hammerstad, Marta; Shi, Chunlin & Meng, Sida [Show all 9 contributors for this article] (2025). SlIDL6–SlHSL1/2/3 ligand-receptor pairs regulate tomato pedicel abscission. Journal of Integrative Agriculture (JIA). ISSN 2095-3119. 25(1), p. 118–126. doi: 10.1016/j.jia.2025.03.018. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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 [Show all 11 contributors for this article] (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. Full text in Research Archive Show summary

  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 [Show all 8 contributors for this article] (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. Full text in Research Archive Show summary

  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), p. 181–193. doi: 10.1107/S205979832400113X. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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), p. 3019–3031. doi: 10.1002/2211-5463.13289. Full text in Research Archive Show summary

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

• Furumizu, Chihiro; Krabberød, Anders Kristian; Hammerstad, Marta; Alling, Renate; Wildhagen, Mari & Sawa, Shinichiro [Show all 7 contributors for this article] (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), p. 2915–2934. doi: 10.1093/plcell/koab173. Full text in Research Archive Show summary

  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 [Show all 10 contributors for this article] (2021). Coumarin-Based Triapine Derivatives and Their Copper(II) Complexes: Synthesis, Cytotoxicity and mR2 RNR Inhibition Activity. Biomolecules. 11(862). doi: 10.3390/biom11060862. Full text in Research Archive Show summary

  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 [Show all 14 contributors for this article] (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, p. 11297–11319. doi: 10.1021/acs.inorgchem.1c01275. Full text in Research Archive Show summary

  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, p. 1–19. doi: 10.1016/j.abb.2021.108826. Full text in Research Archive Show summary

  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), p. 4793–4798. doi: 10.1021/acs.biochem.0c00745. Full text in Research Archive Show summary

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

• 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), p. 318–332. doi: 10.1002/bmb.21218. Full text in Research Archive Show summary

  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), p. 1–15. doi: 10.3390/plants8070225. Full text in Research Archive Show summary

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

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

• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST). Bio-protocol. 7(8), p. e2223–e2223. doi: 10.21769/BioProtoc.2223. Full text in Research Archive Show summary

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

• Zaltariov, Mirela F.; Hammerstad, Marta; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W. & Cazacu, Maria [Show all 13 contributors for this article] (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, p. 3532–3549. doi: 10.1021/acs.inorgchem.6b03178. Full text in Research Archive Show summary

  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), p. 4998–5001. doi: 10.1021/acs.biochem.6b00699. Full text in Research Archive

View all works in NVA

• Hammerstad, Marta; Gudim, Ingvild; Andersen, Hilde Kristin; Aracena-Solem, Matias & Hersleth, Hans-Petter (2026). Metals and thiols in bacterial defense and survival. Full text in Research Archive
• Hammerstad, Marta & Hersleth, Hans-Petter (2025). Structural studies of flavodoxins from Bacillus cereus in different redox states. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Gudim, Ingvild; Gutiérrez-Fernández, Javier & Hersleth, Hans-Petter (2025). Structural and functional studies of enzymes involved in thiol-based redox pathways. Full text in Research Archive
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2025). Structural and functional insights into bacillithiol disulfide reductase – A potential antimicrobial target. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Gutiérrez-Fernández, Javier; Gudim, Ingvild & Hersleth, Hans-Petter (2024). Structures of bacilli­thiol and myco­thiol di­sulfide reductases of bacilli and mycobacteria. Acta Crystallographica Section A: Foundations of Crystallography. ISSN 0108-7673. 80. doi: 10.1107/S2053273324099200. Full text in Research Archive
• Hammerstad, Marta; Karlsen, Marthe Lindahl; Fernandez, Javier Gutierrez; Gudim, Ingvild & Hersleth, Hans-Petter (2024). Structures of bacillithiol and mycothiol disulfide reductases of Firmicutes and Actinobacteria. Full text in Research Archive
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2024). Structural and functional investigation of bacillithiol disulfide reductases. Full text in Research Archive
• 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. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Karlsen, Marthe Lindahl; Hersleth, Hans-Petter & Hammerstad, Marta (2023). Diversity among bacillithiol disulfide reductases – a structural and functional investigation. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2023). Structural determination of Mycobacterium smegmatis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2023). A Research-inspired Laboratory Module on a Redox Protein Combining Biochemistry and Structural Biology. Full text in Research Archive
• Fernandez, Javier Gutierrez; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Structural determination of Mycobacterium tuberculosis and Rhodococcus erythropolis mycothiol disulphide reductases. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Full text in Research Archive
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function. Full text in Research Archive
• 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. Full text in Research Archive
• Rese, Morten; Hammerstad, Marta & Hersleth, Hans-Petter (2022). Diversity in myoglobin function. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2022). Redox protection of pathogens by low molecular weight thiols. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild & Hersleth, Hans-Petter (2020). The Crystal Structures of Bacillithiol Disulfide Reductase YpdA Reveal Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductases. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Enzyme Activation by a Flavoprotein Redox Network. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Beek, Wouter van & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins. Acta Crystallographica Section A: Foundations and Advances. A75, p. e123–e123. doi: 10.1107/S2053273319094336. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Enzyme activation by a flavoprotein redox network. Acta Crystallographica Section A: Foundations and Advances. A75, p. e128–e128. doi: 10.1107/S2053273319094282. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2019). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Beek, Wouter van & Hersleth, Hans-Petter (2019). New structural insight into the well known peptide flip observed in flavodoxins. Full text in Research Archive
• Hammerstad, Marta; Zaltariov, Mirela F.; Arabshahi, Homayon John; Jovanovic, Katarina; Richter, Klaus W. & Cazacu, Maria [Show all 14 contributors for this article] (2019). New thiosemicarbazone derivatives and their copper(II) complexes as potential inhibitors against mammalian ribonucleotide reductase. Full text in Research Archive
• 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. Full text in Research Archive
• Hammerstad, Marta; Gudim, Ingvild; Lofstad, Marie; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2019). Characterization of Proteins in the Ribonucleotide Reductase RedoxNetwork. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Røhr, Åsmund Kjendseth & Hersleth, Hans-Petter (2018). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus. Full text in Research Archive
• 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. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2018). Enzyme activation by a flavoprotein redox network. NBS-nytt. ISSN 0801-3535. p. 40–40. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Enzyme activation by a flavoprotein redox network. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus. Full text in Research Archive
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III in Bacillus cereus. Full text in Research Archive
• Shoor, Marita; Gudim, Ingvild; Hammerstad, Marta & Hersleth, Hans-Petter (2017). Structural and functional characterization of redox proteins in an enzyme activating network involving the thioredoxin reductase in Bacillus cereus. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib RNR by a flavodoxin reductase in B. cereus. NSB-nytt. p. 105–105. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the Class Ib Ribonucleotide Reductase by a Flavin Network in Bacillus cereus. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class IB ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus. Full text in Research Archive
• Johannesen, Hedda; Hammerstad, Marta; Hersleth, Hans-Petter & Andersson, K. Kristoffer (2017). A structural and functional investigation of ribonucleotide reductase class III. Full text in Research Archive
• 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. p. 63–63. Full text in Research Archive
• Zaltariov, Mirela-Fernanda; Hammerstad, Marta; Arabshahi, Homayon John; Jovanović, Katarina; Richter, Klaus W. & Cazacu, Maria [Show all 13 contributors for this article] (2017). New iminodiacetate-thiosemicarbazone hybrids and their copper(II) complexes are potential mR2 RNR inhibitors with high antiproliferative activity. NBS-nytt. ISSN 0801-3535. p. 104–104. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2017). Activation of the class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus. Full text in Research Archive
• Hammerstad, Marta; Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter & Zaltariov, Mirela-Fernanda [Show all 9 contributors for this article] (2017). INHIBITION OF CLASS 1A RIBONUCLEOTIDE REDUCTASE, AND A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE. Full text in Research Archive Show summary

  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. Full text in Research Archive
• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I. [Show all 7 contributors for this article] (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. p. 45–45. Full text in Research Archive
• Hammerstad, Marta; Hersleth, Hans-Petter; Lofstad, Marie; Johannesen, Hedda; Tomter, Ane Berg & Røhr, Åsmund Kjendseth [Show all 7 contributors for this article] (2016). Structural Insight into the Function of Ribonucleotide Reductase. Full text in Research Archive
• Johannesen, Hedda; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2016). A Structural and Functional Investigation of Ribonucleotide Reductase Class III In Bacillus Cereus. Full text in Research Archive
• Lofstad, Marie; Gudim, Ingvild; Kjendseth, Åsmund Røhr; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2016). Activation of Class Ib Ribonucleotide Reductase by NRDI and Its Reductase Partner in Bacillus cereus. Full text in Research Archive
• 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. Full text in Research Archive
• Lofstad, Marie; Böttger, Lars H.; Kjendseth, Åsmund Røhr; Hersleth, Hans-Petter; Hammerstad, Marta & Solomon, Edward I. [Show all 7 contributors for this article] (2016). A COMPARISON OF THE DIMANGANESE ACTIVE SITES OF CLASS IB RIBONUCLEOTIDE REDUCTASE AND MANGANESE CATALASE BY CD AND MCD SPECTROSCOPY. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Hammerstad, Marta; Kjendseth, Åsmund Røhr & Hersleth, Hans-Petter (2016). Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• 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. p. 65–65. Full text in Research Archive
• 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. p. 64–64. Full text in Research Archive
• Gudim, Ingvild; Lofstad, Marie; Andersson, K. Kristoffer; Hammerstad, Marta & Hersleth, Hans-Petter (2016). Probing enzyme activation networks ‐ structural and functional studies of flavoproteins in Bacillus cereus. NBS-nytt. ISSN 0801-3535. p. 45–45. Full text in Research Archive
• Lofstad, Marie; Böttger, Lars H.; Røhr, Åsmund Kjendseth; Hammerstad, Marta; Hersleth, Hans-Petter & Solomon, Edward I. [Show all 7 contributors for this article] (2015). A comparison of the dimanganese active sites of class Ib ribonucleotide reductase and manganese catalase by CD and MCD spectroscopy. Full text in Research Archive
• Monka, Susanne; Hammerstad, Marta; Andersson, K. Kristoffer & Hersleth, Hans-Petter (2015). On the way to determine the structure of a ferredoxin in B. cereus. Full text in Research Archive Show summary

  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. p. 97–97. Full text in Research Archive Show summary

  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. Full text in Research Archive
• 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. p. 47–47. Full text in Research Archive Show summary

  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. Full text in Research Archive
• 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. Full text in Research Archive
• 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, p. C434–C434. Full text in Research Archive Show summary

  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. p. 85–85. Full text in Research Archive
• 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, p. C1657–C1657. Full text in Research Archive Show summary

  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. Full text in Research Archive
• 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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. Full text in Research Archive
• 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. Full text in Research Archive
• Gudim, Ingvild; Hersleth, Hans-Petter; Hammerstad, Marta & Sørlie, Morten (2018). Characterisation of flavodoxin and ferredoxin/flavodoxin reductases from Bacillus cereus and their interactions. Reprosentralen, University of Oslo. Full text in Research Archive
• Shoor, Marita; Hersleth, Hans-Petter; Gudim, Ingvild & Hammerstad, Marta (2017). Structural and functional characterization of the redox protein Thioredoxin reductase from Bacillus cereus. Universitetet i Oslo. Full text in Research Archive
• 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. Full text in Research Archive
• Wu, Bernt; Hersleth, Hans-Petter; Hammerstad, Marta & Andersson, K. Kristoffer (2015). Purification and characterization of Flavohemoglobin - A flavoheme enzyme. Universitetet i Oslo. Full text in Research Archive Show summary

  Flavohemoglobin is a flavoheme enzyme that catalyzes dioxygenation of nitric oxide (NO) to nitrate NO3–. 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. Full text in Research Archive Show summary

  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. Full text in Research Archive

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