| 1. |
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| 2. |
- Braun, Tatjana, et al.
(författare)
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Archaeal actin from a hyperthermophile forms a single-stranded filament
- 2015
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:30, s. 9340-9345
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Tidskriftsartikel (refereegranskat)abstract
- The prokaryotic origins of the actin cytoskeleton have been firmly established, but it has become clear that the bacterial actins form a wide variety of different filaments, different both from each other and from eukaryotic F-actin. We have used electron cryomicroscopy (cryo-EM) to examine the filaments formed by the protein crenactin (a crenarchaeal actin) from Pyrobaculum calidifontis, an organism that grows optimally at 90 degrees C. Although this protein only has similar to 20% sequence identity with eukaryotic actin, phylogenetic analyses have placed it much closer to eukaryotic actin than any of the bacterial homologs. It has been assumed that the crenactin filament is double-stranded, like F-actin, in part because it would be hard to imagine how a single-stranded filament would be stable at such high temperatures. We show that not only is the crenactin filament single-stranded, but that it is remarkably similar to each of the two strands in F-actin. A large insertion in the crenactin sequence would prevent the formation of an F-actin-like double-stranded filament. Further, analysis of two existing crystal structures reveals six different subunit-subunit interfaces that are filament-like, but each is different from the others in terms of significant rotations. This variability in the subunit-subunit interface, seen at atomic resolution in crystals, can explain the large variability in the crenactin filaments observed by cryo-EM and helps to explain the variability in twist that has been observed for eukaryotic actin filaments.
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| 3. |
- Cicero, Giancarlo, et al.
(författare)
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Study of the oxidative half-reaction catalyzed by a non-heme ferrous catalytic center by means of structural and computational methodologies
- 2007
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Ingår i: International Journal of Quantum Chemistry. - : Wiley. - 0020-7608 .- 1097-461X. ; 107:6, s. 1514-1522
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Tidskriftsartikel (refereegranskat)abstract
- Deacetoxycephalosporin C synthase (DAOCS) is a mononuclear ferrous enzyme that catalyzes the expansion of the five-membered thiazolidine ring of the penicillin nucleus into the six-membered dihydrothiazine ring of the cephalosporins. In the first half-reaction with dioxygen and 2-oxoglutarate, a reactive iron-oxygen species is produced that can subsequently react with the penicillin substrate to yield the cephalosporin. We describe quantum mechanical calculations of the first part of the reaction based on the high-resolution structures of the active site of DAOCS and its complexes with ligands. These studies are aimed at understanding how the reactive species can be produced and contained in the active site of the enzyme. The results demonstrate the priming of the active site by the co-substrate for oxygen binding and hint to the presence of a stable iron-peroxo intermediate in equilibrium with a more reactive ferryl species and the formation of CO2 as a leaving group by decarboxylation of 2-oxoglutarate. A conclusion from these studies is that substitution of CO2 by the penicillin substrate triggers the oxidation reaction in a booby-trap-like mechanism.
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| 4. |
- Hasse, Dirk, et al.
(författare)
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Structure and mechanism of piperideine-6-carboxylate dehydrogenase from Streptomyces clavuligerus
- 2019
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Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 75, s. 1107-1118
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Tidskriftsartikel (refereegranskat)abstract
- The core of beta-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. beta-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid alpha-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form alpha-aminoadipate semialdehyde) to alpha-aminoadipic acid, is catalysed by the NAD(+)-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD(+), the product alpha-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD(+) binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.
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| 5. |
- Larsson, Anna M., et al.
(författare)
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Crystal structures of β-carboxysome shell protein CcmP : ligand binding correlates with the closed or open central pore
- 2017
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Ingår i: Journal of Experimental Botany. - : OXFORD UNIV PRESS. - 0022-0957 .- 1460-2431. ; 68:14, s. 3857-3867
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacterial CO2 fixation is promoted by encapsulating and co-localizing the CO2-fixing enzymes within a protein shell, the carboxysome. A key feature of the carboxysome is its ability to control selectively the flux of metabolites in and out of the shell. The beta-carboxysome shell protein CcmP has been shown to form a double layer of pseudohexamers with a relatively large central pore (similar to 13 angstrom diameter), which may allow passage of larger metabolites such as the substrate for CO2 fixation, ribulose 1,5-bisphosphate, through the shell. Here we describe two crystal structures, at 1.45 angstrom and 1.65 angstrom resolution, of CcmP from Synechococcus elongatus PCC7942 (SeCcmP). The central pore of CcmP is open or closed at its ends, depending on the conformation of two conserved residues, Glu69 and Arg70. The presence of glycerol resulted in a pore that is open at one end and closed at the opposite end. When glycerol was omitted, both ends of the barrel became closed. A binding pocket at the interior of the barrel featured residual density with distinct differences in size and shape depending on the conformation, open or closed, of the central pore of SeCcmP, suggestive of a metabolite-driven mechanism for the gating of the pore.
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| 6. |
- Lejon, Sara, 1978-
(författare)
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Evasion and Attack: Structural Studies of a Bacterial Albumin-binding Protein and of a Cephalosporin Biosynthetic Enzyme
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule.Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.
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| 7. |
- Lejon, Sara, et al.
(författare)
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The last step in cephalosporin C formation revealed : Crystal Structures of Deacetylcephalosporin C Acetyltransferase from Acremonium chrysogenum in Complexes with Reaction Intermediates
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 377:3, s. 935-944
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Tidskriftsartikel (refereegranskat)abstract
- Deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of cephalosporin C, a broad-spectrum beta-lactam antibiotic of large clinical importance. The acetyl transfer step has been suggested to be limiting for cephalosporin C biosynthesis, but has so far escaped detailed structural analysis. We present here the crystal structures of DAC-AT in complexes with reaction intermediates, providing crystallographic snapshots of the reaction mechanism. The enzyme is found to belong to the alpha/beta hydrolase class of acetyltransferases, and the structures support previous observations of a double displacement mechanism for the acetyl transfer reaction in other members of this class of enzymes. The structures of DAC-AT reported here provide evidence of a stable acyl - enzyme complex, thus underpinning a mechanism involving acetylation of a catalytic serine residue by acetyl coenzyme A, followed by transfer of the acetyl group to deacetylcephalosporin C through a suggested tetrahedral transition state.
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| 8. |
- Lemmens, Liesbeth, et al.
(författare)
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YtrA(Sa), a GntR-Family Transcription Factor, Represses Two Genetic Loci Encoding Membrane Proteins in Sulfolobus acidocaldarius
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- In bacteria, the GntR family is a widespread family of transcription factors responsible for the regulation of a myriad of biological processes. In contrast, despite their occurrence in archaea only a little information is available on the function of GntR-like transcription factors in this domain of life. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius harbors a GntR-like regulator belonging to the YtrA subfamily, encoded as the first gene in an operon with a second gene encoding a putative membrane protein. Here, we present a detailed characterization of this regulator, named YtrA(Sa), with a focus on regulon determination and mechanistic analysis with regards to DNA binding. Genome-wide chromatin immunoprecipitation and transcriptome experiments, the latter employing a ytrA(Sa) overexpression strain, demonstrate that the regulator acts as a repressor on a very restricted regulon, consisting of only two targets including the operon encoding its own gene and a distinct genetic locus encoding another putative membrane protein. For both targets, a conserved 14-bp semi-palindromic binding motif was delineated that covers the transcriptional start site and that is surrounded by additional half-site motifs. The crystallographic structure of YtrA(Sa) was determined, revealing a compact dimeric structure in which the DNA-binding motifs are oriented ideally to enable a specific high-affinity interaction with the core binding motif. This study provides new insights into the functioning of a YtrA-like regulator in the archaeal domain of life.
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| 9. |
- Lindås, Ann-Christin, et al.
(författare)
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Archaeal Actin-Family Filament Systems.
- 2017
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Ingår i: Sub-cellular biochemistry. - Cham : Springer International Publishing. - 0306-0225. ; 84, s. 379-392
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Tidskriftsartikel (refereegranskat)abstract
- Actin represents one of the most abundant and conserved eukaryotic proteins over time, and has an important role in many different cellular processes such as cell shape determination, motility, force generation, cytokinesis, amongst many others. Eukaryotic actin has been studied for decades and was for a long time considered a eukaryote-specific trait. However, in the early 2000s a bacterial actin homolog, MreB, was identified, characterized and found to have a cytoskeletal function and group within the superfamily of actin proteins. More recently, an actin cytoskeleton was also identified in archaea. The genome of the hyperthermophilic crenarchaeon Pyrobaculum calidifontis contains a five-gene cluster named Arcade encoding for an actin homolog, Crenactin, polymerizing into helical filaments spanning the whole length of the cell. Phylogenetic and structural studies place Crenactin closer to the eukaryotic actin than to the bacterial homologues. A significant difference, however, is that Crenactin can form single helical filaments in addition to filaments containing two intertwined proto filaments. The genome of the recently discovered Lokiarchaeota encodes several different actin homologues, termed Lokiactins, which are even more closely related to the eukaryotic actin than Crenactin. A primitive, dynamic actin-based cytoskeleton in archaea could have enabled the engulfment of the alphaproteobacterial progenitor of the mitochondria, a key-event in the evolution of eukaryotes.
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| 10. |
- Lindås, Ann-Christin, et al.
(författare)
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Structure of crenactin, an archaeal actin homologue active at 90 degrees C
- 2014
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 70, s. 492-500
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of the archaeal actin, crenactin, from the rod-shaped hyperthermophilic (optimal growth at 90 degrees C) crenarchaeon Pyrobaculum calidifontis is reported at 3.35 angstrom resolution. Despite low amino-acid sequence identity, the three-dimensional structure of the protein monomer is highly similar to those of eukaryotic actin and the bacterial MreB protein. Crenactin-specific features are also evident, as well as elements that are shared between crenactin and eukaryotic actin but are not found in MreB. In the crystal, crenactin monomers form right-handed helices, demonstrating that the protein is capable of forming filament-like structures. Monomer interactions in the helix, as well as interactions between crenactin and ADP in the nucleotide-binding pocket, are resolved at the atomic level and compared with those of actin and MreB. The results provide insights into the structural and functional properties of a heat-stable archaeal actin and contribute to the understanding of the evolution of actin-family proteins in the three domains of life.
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