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Rozman Grinberg, I., Berglund, S., Hasan, M., Lundin, D., Ho, F. M., Magnuson, A., . . . Berggren, G. (2019). Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading. Journal of Biological Inorganic Chemistry, 24(6), 863-877
Open this publication in new window or tab >>Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading
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2019 (English)In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, no 6, p. 863-877Article in journal (Refereed) Published
Abstract [en]

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O 2 ·− ). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.

Keywords
Ribonucleotide reductase, Dimanganese cofactor, Radicals, Electron paramagnetic resonance, X-ray crystallography, Phylogeny
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-392170 (URN)10.1007/s00775-019-01697-8 (DOI)000487094500011 ()31414238 (PubMedID)
Funder
EU, European Research Council, 714102Swedish Research Council, 621-2014-5670Swedish Research Council, 2016-04855Swedish Research Council, 2016-01920Swedish Cancer Society, 2018/820Magnus Bergvall FoundationWenner-Gren Foundations
Available from: 2019-08-30 Created: 2019-08-30 Last updated: 2019-10-30Bibliographically approved
Magnuson, A. (2019). Heterocyst Thylakoid Bioenergetics. LIFE-BASEL, 9(1), Article ID 13.
Open this publication in new window or tab >>Heterocyst Thylakoid Bioenergetics
2019 (English)In: LIFE-BASEL, ISSN 2075-1729, Vol. 9, no 1, article id 13Article, review/survey (Refereed) Published
Abstract [en]

Heterocysts are specialized cells that differentiate in the filaments of heterocystous cyanobacteria. Their role is to maintain a microoxic environment for the nitrogenase enzyme during diazotrophic growth. The lack of photosynthetic water oxidation in the heterocyst puts special constraints on the energetics for nitrogen fixation, and the electron transport pathways of heterocyst thylakoids are slightly different from those in vegetative cells. During recent years, there has been a growing interest in utilizing heterocysts as cell factories for the production of fuels and other chemical commodities. Optimization of these production systems requires some consideration of the bioenergetics behind nitrogen fixation. In this overview, we emphasize the role of photosynthetic electron transport in providing ATP and reductants to the nitrogenase enzyme, and provide some examples where heterocysts have been used as production facilities.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
cyanobacteria, thylakoid, photosynthesis nitrogen fixation, ferredoxin, biofuel
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-382801 (URN)10.3390/life9010013 (DOI)000464137000001 ()30691012 (PubMedID)
Available from: 2019-05-03 Created: 2019-05-03 Last updated: 2019-05-03Bibliographically approved
Li, X., Mustila, H., Magnuson, A. & Stensjö, K. (2018). Homologous overexpression of NpDps2 and NpDps5 increases the tolerance for oxidative stress in the multicellular cyanobacterium Nostoc punctiforme. FEMS Microbiology Letters, 365(18), Article ID fny198.
Open this publication in new window or tab >>Homologous overexpression of NpDps2 and NpDps5 increases the tolerance for oxidative stress in the multicellular cyanobacterium Nostoc punctiforme
2018 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 365, no 18, article id fny198Article in journal (Refereed) Published
Abstract [en]

The filamentous cyanobacterium Nostoc punctiforme has several oxidative stress-managing systems, including Dps proteins. Dps proteins belong to the ferritin superfamily and are involved in abiotic stress management in prokaryotes. Previously, we found that one of the five Dps proteins in N. punctiforme, NpDps2, was critical for H2O2 tolerance. Stress induced by high light intensities is aggravated in N. punctiforme strains deficient of either NpDps2, or the bacterioferritin-like NpDps5. Here, we have investigated the capacity of NpDps2 and NpDps5 to enhance stress tolerance by homologous overexpression of these two proteins in N. punctiforme. Both overexpression strains were found to tolerate twice as high concentrations of added H2O2 as the control strain, indicating that overexpression of either NpDps2 or NpDps5 will enhance the capacity for H2O2 tolerance. Under high light intensities, the overexpression of the two NpDps did not enhance the tolerance against general light-induced stress. However, overexpression of the heterocyst-specific NpDps5 in all cells of the filament led to a higher amount of chlorophyll-binding proteins per cell during diazotrophic growth. The OENpDps5 strain also showed an increased tolerance to ammonium-induced oxidative stress. Our results provide information of how Dps proteins may be utilised for engineering of cyanobacteria with enhanced stress tolerance.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-365085 (URN)10.1093/femsle/fny198 (DOI)000449462200012 ()30107525 (PubMedID)
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2019-01-22Bibliographically approved
Magnuson, A., Raleiras, P., Meszaros, L. S., Khanna, N., Miranda, H., Ho, F. M., . . . Styring, S. (2018). Sustainable photobiological hydrogen production via protein engineering of cyanobacterial hydrogenases. Paper presented at 255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water, MAR 18-22, 2018, New Orleans, USA.. Abstract of Papers of the American Chemical Society, 255
Open this publication in new window or tab >>Sustainable photobiological hydrogen production via protein engineering of cyanobacterial hydrogenases
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2018 (English)In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 255Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Washington, D.C.: American Chemical Society (ACS), 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-368928 (URN)000435539900440 ()
Conference
255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water, MAR 18-22, 2018, New Orleans, USA.
Note

Meeting Abstract: 443

Available from: 2018-12-11 Created: 2018-12-11 Last updated: 2018-12-11Bibliographically approved
Moparthi, V., Li, X., Vavitsas, K., Dzhygyr, I., Sandh, G., Magnuson, A. & Stensjö, K. (2016). The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme. Biochimica et Biophysica Acta - Bioenergetics, 1857, 1766-1776
Open this publication in new window or tab >>The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme
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2016 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1857, p. 1766-1776Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiologica characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance,H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.

Place, publisher, year, edition, pages
Elsevier, 2016
Keywords
adaptation; cyanobacteria; ferritin; photosystem; light-stress; ROS
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-301875 (URN)10.1016/j.bbabio.2016.08.003 (DOI)000384867400006 ()
Funder
Swedish Energy Agency, 11674-5Knut and Alice Wallenberg Foundation, 2011.0067Carl Tryggers foundation Lars Hierta Memorial Foundation
Available from: 2016-08-25 Created: 2016-08-25 Last updated: 2018-03-01Bibliographically approved
Magnuson, A. & Cardona, T. (2016). Thylakoid membrane function in heterocysts. Biochimica et Biophysica Acta - Bioenergetics, 1857(3), 309-319
Open this publication in new window or tab >>Thylakoid membrane function in heterocysts
2016 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1857, no 3, p. 309-319Article, review/survey (Refereed) Published
Abstract [en]

Multicellular cyanobacteria form different cell types in response to environmental stimuli. Under nitrogen limiting conditions a fraction of the vegetative cells in the filament differentiate into heterocysts. Heterocysts are specialized in atmospheric nitrogen fixation and differentiation involves drastic morphological changes on the cellular level, such as reorganization of the thylakoid membranes and differential expression of thylakoid membrane proteins. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase by developing an extra polysaccharide layer that limits air diffusion into the heterocyst and by upregulating heterocyst-specific respiratory enzymes. In this review article, we summarize what is known about the thylakoid membrane in heterocysts and compare its function with that of the vegetative cells. We emphasize the role of photosynthetic electron transport in providing the required amounts of ATP and reductants to the nitrogenase enzyme. In the light of recent high-throughput proteomic and transcriptomic data, as well as recently discovered electron transfer pathways in cyanobacteria, our aim is to broaden current views of the bioenergetics of heterocysts. This article is part of a Special Issue entitled: Bioenergetic systems in bacteria.

Place, publisher, year, edition, pages
Elsevier, 2016
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-271309 (URN)10.1016/j.bbabio.2015.10.016 (DOI)000370895500013 ()26545609 (PubMedID)
Funder
Swedish Energy Agency, 11674-5Knut and Alice Wallenberg Foundation, 2011.0067
Available from: 2016-01-07 Created: 2016-01-07 Last updated: 2019-02-11Bibliographically approved
Raleiras, P., Khanna, N., Miranda, H., Meszaros, L. S., Krassen, H., Ho, F., . . . Styring, S. (2016). Turning around the electron flow in an uptake hydrogenase. EPR spectroscopy and in vivo activity of a designed mutant in HupSL from Nostoc punctiforme. Energy & Environmental Science, 9(2), 581-594
Open this publication in new window or tab >>Turning around the electron flow in an uptake hydrogenase. EPR spectroscopy and in vivo activity of a designed mutant in HupSL from Nostoc punctiforme
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2016 (English)In: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 9, no 2, p. 581-594Article in journal (Refereed) Published
Abstract [en]

The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 produces hydrogen via nitrogenase in heterocysts upon onset of nitrogen-fixing conditions. N. punctiforme expresses concomitantly the uptake hydrogenase HupSL, which oxidizes hydrogen in an effort to recover some of the reducing power used up by nitrogenase. Eliminating uptake activity has been employed as a strategy for net hydrogen production in N. punctiforme (Lindberg et al., Int. J. Hydrogen Energy, 2002, 27, 1291-1296). However, nitrogenase activity wanes within a few days. In the present work, we modify the proximal iron-sulfur cluster in the hydrogenase small subunit HupS by introducing the designed mutation C12P in the fusion protein f-HupS for expression in E. coli (Raleiras et al., J. Biol. Chem., 2013, 288, 18345-18352), and in the full HupSL enzyme for expression in N. punctiforme. C12P f-HupS was investigated by EPR spectroscopy and found to form a new paramagnetic species at the proximal cluster site consistent with a [4Fe-4S] to [3Fe-4S] cluster conversion. The new cluster has the features of an unprecedented mixed-coordination [3Fe-4S] metal center. The mutation was found to produce stable protein in vitro, in silico and in vivo. When C12P HupSL was expressed in N. punctiforme, the strain had a consistently higher hydrogen production than the background [capital Delta]hupSL mutant. We conclude that the increase in hydrogen production is due to the modification of the proximal iron-sulfur cluster in HupS, leading to a turn of the electron flow in the enzyme.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2016
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-271302 (URN)10.1039/C5EE02694F (DOI)000369744500023 ()
Funder
Knut and Alice Wallenberg Foundation, 2011.0067EU, FP7, Seventh Framework Programme, 317184Swedish Energy Agency, 11674-5
Available from: 2016-01-07 Created: 2016-01-07 Last updated: 2017-12-01Bibliographically approved
Wennman, A., Jernerén, F., Magnuson, A. & Oliw, E. H. (2015). Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid. Archives of Biochemistry and Biophysics, 583, 87-95
Open this publication in new window or tab >>Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid
2015 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 583, p. 87-95Article in journal (Refereed) Published
Abstract [en]

Magnaporthe oryzae causes rice blast disease and has become a model organism of fungal infections. M. oryzae can oxygenate fatty acids by 7,8-linoleate diol synthase, 10R-dioxygenase-epoxy alcohol synthase, and by a putative manganese lipoxygenase (Mo-MnLOX). The latter two are transcribed during infection. The open reading frame of Mo-MnLOX was deduced from genome and cDNA analysis. Recombinant Mo-MnLOX was expressed in Pichia pastoris and purified to homogeneity. The enzyme contained protein-bound Mn and oxidized 18:2n-6 and 18:3n-3 to 9S-, 11-, and 13R-hydroperoxy metabolites by suprafacial hydrogen abstraction and oxygenation. The 11-hydroperoxides were subject to β-fragmentation with formation of 9S- and 13R-hydroperoxy fatty acids. Oxygen consumption indicated apparent kcat values of 2.8 s(-1) (18:2n-6) and 3.9 s(-1) (18:3n-3), and UV analysis yielded apparent Km values of 8 and 12 μM, respectively, for biosynthesis of cis-trans conjugated metabolites. 9S-Hydroperoxy-10E,12Z,15Z-octadecatrienoic acid was rapidly further oxidized to a triene, 9S,16S-dihydroperoxy-10E,12Z,14E-octadecatrienoic acid. In conclusion, we have expressed, purified and characterized a new MnLOX from M. oryzae. The pathogen likely secretes Mo-MnLOX and phospholipases to generate oxylipins and to oxidize lipid membranes of rice cells and the cuticle.

Keywords
Fusarium gloeosporioides, gene expression, oxygenation mechanism, oxylipins, Pichia pastoris, yeast expression, mass spectrometry, Fusarium oxysporum
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-262345 (URN)10.1016/j.abb.2015.07.014 (DOI)000361644400011 ()26264916 (PubMedID)
Funder
Swedish Research Council, 03X-06523Knut and Alice Wallenberg Foundation, KAW 2004.0123Swedish Energy Agency
Available from: 2015-09-14 Created: 2015-09-14 Last updated: 2019-09-11
Wennman, A., Magnuson, A., Hamberg, M. & Oliw, E. H. (2015). Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers. Journal of Lipid Research, 56(8), 1606-1615
Open this publication in new window or tab >>Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers
2015 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 56, no 8, p. 1606-1615Article in journal (Refereed) Published
Abstract [en]

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (Fox-LOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11 R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid beta-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-H-2] 18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the H-2-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero3- phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18: 2n-6 to the same major products as Cg-MnLOX.Jlr Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the pro R rather than the proShydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.

Keywords
Fusarium gloeosporioides, gene expression, oxygenation mechanism, oxylipins, Pichia pastoris, yeast expression, mass spectrometry, Fusarium oxysporum
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-260821 (URN)10.1194/jlr.M060178 (DOI)000358666000022 ()26113537 (PubMedID)
Funder
Swedish Research Council, 03X-06523Knut and Alice Wallenberg Foundation, KAW 2004.0123
Available from: 2015-08-27 Created: 2015-08-25 Last updated: 2017-12-04Bibliographically approved
Raleiras, P., Hammarström, L., Lindblad, P., Styring, S. & Magnuson, A. (2015). Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme. Journal of Inorganic Biochemistry, 148, 57-61
Open this publication in new window or tab >>Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme
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2015 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 148, p. 57-61Article in journal (Refereed) Published
Abstract [en]

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-253857 (URN)10.1016/j.jinorgbio.2015.03.018 (DOI)000357702100008 ()25912316 (PubMedID)
Available from: 2015-06-03 Created: 2015-06-03 Last updated: 2017-12-04Bibliographically approved
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