Inger Andersson

Uppsala University, Uppsala, Uppsala, Sweden

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Publications (71)529.95 Total impact

  • Dirk Hasse · Anna M Larsson · Inger Andersson
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    ABSTRACT: The CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inactivated by the formation of dead-end complexes with inhibitory sugar phosphates. In plants and green algae, the ATP-dependent motor protein Rubisco activase restores catalytic competence by facilitating conformational changes in Rubisco that promote the release of the inhibitory compounds from the active site. Here, the crystal structure of Rubisco activase from Arabidopsis thaliana is presented at 2.9 Å resolution. The structure reveals an AAA+ two-domain structure. More than 100 residues in the protein were not visible in the electron-density map owing to conformational disorder, but were verified to be present in the crystal by mass spectrometry. Two sulfate ions were found in the structure. One was bound in the loop formed by the Walker A motif at the interface of the domains. A second sulfate ion was bound at the N-terminal end of the first helix of the C-terminal domain. The protein packs in a helical fashion in the crystal, as observed previously for Rubisco activase, but differences in the helical pitch indicate flexibility in the packing of the protein.
    No preview · Article · Apr 2015 · Acta Crystallographica Section D Biological Crystallography
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    ABSTRACT: We present a proof-of-concept three-dimensional reconstruction of the giant mimivirus particle from experimentally measured diffraction patterns from an x-ray free-electron laser. Three-dimensional imaging requires the assembly of many two-dimensional patterns into an internally consistent Fourier volume. Since each particle is randomly oriented when exposed to the x-ray pulse, relative orientations have to be retrieved from the diffraction data alone. We achieve this with a modified version of the expand, maximize and compress algorithm and validate our result using new methods.
    No preview · Article · Mar 2015 · Physical Review Letters
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    ABSTRACT: There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.
    No preview · Article · Feb 2015 · Nature Communications
  • I. Andersson · K. Valegård
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    ABSTRACT: Central steps in the biosynthetic pathways of some of the most commonly used antibiotics, the cephalosporins, are catalysed by 2-oxoglutarate (2OG)-dependent oxygenases. Deacetoxycephalosporin C synthase (DAOCS) catalyses the 2OG-dependent oxidative expansion of the five-membered thiazolidine ring of the penicillin nucleus into the six-membered dihydrothiazine ring of the cephalosporin nucleus. DAOCS uses dioxygen to create a reactive iron-oxygen intermediate from ferrous ion to drive the reaction. In prokaryotic cephalosporin producers, the cephalosporin product, DAOC, is hydroxylated at the 3′-position to form deacetylcephalosporin C (DAC) as catalysed by a second 2OG-dependent enzyme, DAC synthase (DACS). In eukaryotic cephalosporin producers, the reaction is catalysed by a bifunctional enzyme, DAOC/DACS, that catalyses both the ring expansion and the 3′-hydroxylation reactions. The prokaryotic and eukaryotic enzymes are closely related to DAOCS by sequence, suggesting these enzymes may have evolved by gene duplication. Cephamycin C-producing microorganisms use two enzymes, encoded by the genes cmcI/J, to convert cephalosporins to their 7α-methoxy derivatives that are less vulnerable to β-lactam hydrolysing enzymes. The methoxylation reaction is dependent on Fe(ii), 2OG and S-adenosylmethionine, suggesting the involvement of another 2OG-dependent oxygenase. Herein, structural and mechanistic features are summarized for these 2OG enzymes that utilize this common and flexible mode of dioxygen activation.
    No preview · Chapter · Jan 2015
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    ABSTRACT: We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study
    No preview · Article · Dec 2014 · Nature Photonics
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    ABSTRACT: Protein-gas interactions are important in biology. The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses two competing reactions involving CO2 and O2 as substrates. Carboxylation of the common substrate ribulose-1,5-bisphosphate leads to photosynthetic carbon assimilation, while the oxygenation reaction competes with carboxylation and reduces photosynthetic productivity. The migration of the two gases in and around Rubisco was investigated using molecular dynamics simulations. The results indicate that at equal concentrations of the gases, Rubisco binds CO2 stronger than it does O2. Amino acids with small hydrophobic side chains are the most proficient in attracting CO2, indicating a significant contribution of the hydrophobic effect in the interaction. On average, residues in the small subunit bind approximately twice as much CO2 as do residues in the large subunit. We did not detect any cavities that would provide a route to the active site for the gases. Instead, CO2 appears to be guided toward the active site through a CO2 binding region around the active site opening that extends to the closest neighbouring small subunits. Taken together, these results suggest the small subunit may function as a "reservoir" for CO2 storage.
    No preview · Article · Feb 2014 · Journal of the American Chemical Society
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    ABSTRACT: Glycine decarboxylase, or P-protein, is a pyridoxal 5'-phosphate (PLP) dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an α2 homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apo enzyme shows the C-terminus locked in a closed conformation by a disulphide bond between Cys972 in the C-terminus and Cys353 located in the active site. Presence of the disulphide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys972 and Cys353 by Ser using site-directed mutagenesis have distinctly lower specific activities supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353-972 disulphide releases the C-terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulphide bond that acts to stabilise the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier.
    Full-text · Article · Oct 2013 · Journal of Biological Chemistry
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    ABSTRACT: Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.
    No preview · Article · Aug 2013 · Acta Crystallographica Section D Biological Crystallography
  • Michiel van Lun · David van der Spoel · Inger Andersson
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    ABSTRACT: Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L(2) dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance.
    No preview · Article · Sep 2011 · Journal of Molecular Biology
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    ABSTRACT: Single-particle experiments using X-ray Free Electron Lasers produce more than 10(5) snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one or more particles. Experimental data sets also often contain unintentional contamination with different species. We present an unsupervised method able to sort experimental snapshots without recourse to templates, specific noise models, or user-directed learning. The results show 90% agreement with manual classification.
    Full-text · Article · Aug 2011 · Optics Express
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    ABSTRACT: X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.
    No preview · Article · Feb 2011 · Nature
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    ABSTRACT: X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
    Full-text · Article · Feb 2011 · Nature
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    ABSTRACT: Rubisco, the primary photosynthetic carboxylase, evolved 3–4 billion years ago in an anaerobic, high CO2 atmosphere. The combined effect of low CO2 and high O2 levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO2 and O2, leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C4 photosynthesis largely solves this problem by facilitating a high CO2/O2 ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcatCO2 and lower CO2/O2 specificity (SC/O) is well supported, but the structural basis for the differences between C3 and C4 Rubiscos is not clear. Flaveria (Asteraceae) includes C3, C3–C4 intermediate, and C4 species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C3 to C4 photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C4 Rubiscos from the ancestral C3 species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic “C4” SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus.
    Full-text · Article · Dec 2010 · Molecular Biology and Evolution
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    ABSTRACT: In a flash diffraction experiment, a short and extremely intense x-ray pulse illuminates the sample to obtain a diffraction pattern before the onset of significant radiation damage. The over-sampled diffraction pattern permits phase retrieval by iterative phasing methods. Flash diffractive imaging was first demonstrated on an inorganic test object (Chapman et al 2006 Nat. Phys. 2 839–43). We report here experiments on biological systems where individual cells were imaged, using single, 10–15 fs soft x-ray pulses at 13.5 nm wavelength from the FLASH free-electron laser in Hamburg. Simulations show that the pulse heated the sample to about 160 000 K but not before an interpretable diffraction pattern could be obtained. The reconstructed projection images return the structures of the intact cells. The simulations suggest that the average displacement of ions and atoms in the hottest surface layers remained below 3 Å during the pulse.
    Full-text · Article · Sep 2010 · Journal of Physics B Atomic Molecular and Optical Physics
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    Dirk Hasse · Martin Hagemann · Inger Andersson · Hermann Bauwe
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    ABSTRACT: Glycine decarboxylase, or P-protein, is a major enzyme that is involved in the C(1) metabolism of all organisms and in the photorespiratory pathway of plants and cyanobacteria. The protein from Synechocystis sp. PCC 6803 is a homodimer with a mass of 215 kDa. Recombinant glycine decarboxylase was expressed in Escherichia coli and purified by metal-affinity, ion-exchange and gel-filtration chromatography. Crystals of P-protein that diffracted to a resolution of 2.1 A were obtained using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected from cryocooled crystals using synchrotron radiation. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 96.30, b = 135.81, c = 179.08 A.
    Full-text · Article · Feb 2010 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
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    ABSTRACT: Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 A resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.
    Full-text · Article · Nov 2009 · Journal of Molecular Biology
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    ABSTRACT: Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V(c) (V(max) for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the K(m) for CO(2) and O(2) was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 degrees C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 degrees C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of beta-strand 1 of the alpha/beta-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.
    Full-text · Article · May 2008 · Biochemical Journal
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    Preview · Article · Apr 2008
  • Inger Andersson · Anders Backlund
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    ABSTRACT: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO(2) into the biosphere. At the same time Rubisco is an extremely inefficient catalyst and its carboxylase activity is compromised by an opposing oxygenase activity involving atmospheric O(2). The shortcomings of Rubisco have implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. This review uses the information provided in these structures in a structure-based sequence alignment and discusses Rubisco function in the context of structural variations at all levels--amino acid sequence, fold, tertiary and quaternary structure--with an evolutionary perspective and an emphasis on the structural features of the enzyme that may determine its function as a carboxylase.
    No preview · Article · Apr 2008 · Plant Physiology and Biochemistry
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    Inger Andersson
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    ABSTRACT: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the incorporation of inorganic CO2 into the organic molecules of life. Rubisco is extremely inefficient as a catalyst and its carboxylase activity is compromised by numerous side-reactions including oxygenation of its sugar phosphate substrate by atmospheric O2. The reduction in the catalytic efficiency as a result of these processes has implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Several aspects of Rubisco including its complex biosynthesis and multi-step catalytic reaction are subject to tight control involving light, cellular metabolites, and molecular chaperones. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. These provide a molecular framework for the understanding of these processes at the molecular level.
    Preview · Article · Feb 2008 · Journal of Experimental Botany

Publication Stats

3k Citations
529.95 Total Impact Points

Institutions

  • 2013-2015
    • Uppsala University
      • Department of Cell and Molecular Biology
      Uppsala, Uppsala, Sweden
  • 1989-2011
    • Swedish University of Agricultural Sciences
      • • Department of Molecular Biology
      • • Department of Plant Biology and Forest Genetics
      Uppsala, Uppsala, Sweden
  • 2010
    • University of Rostock
      • Institut für Biowissenschaften (IfBi)
      Rostock, Mecklenburg-Vorpommern, Germany
  • 1996
    • Uppsala Monitoring Centre
      Uppsala, Uppsala, Sweden