| 1. |
- Kutin, Yuri, et al.
(författare)
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Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins
- 2016
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Ingår i: Journal of Inorganic Biochemistry. - : Elsevier BV. - 0162-0134 .- 1873-3344. ; 162, s. 164-177
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Tidskriftsartikel (refereegranskat)abstract
- A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of dass Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is undear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn-II and Fe-II. Using EPR and Mfissbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn-II promotes heterobimetallic cofactor assembly. In the absence of Fe-II, R2c cooperatively binds Mn-II at both metal sites, whereas R2lox does not readily bind Mn-II at either site. Heterometallic cofactor assembly is favored at substoichiometric Feu concentrations in R2lox. Fe-II and Mn-II likely bind to the protein in a stepwise fashion, with Feu binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn-II by Fe-II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn-II/Fe-II concentrations in the host organisms from which they were isolated.
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| 2. |
- Lundin, Daniel, 1965-, et al.
(författare)
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The functional diversity and evolutionary relationships of ferritin-like proteins
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- BackgroundThe ferritin-like proteins are evolutionarily related, as evidenced by the topology oftheir signature metal-binding four-helix bundle. They perform diverse functions suchas iron/oxygen detoxification, iron storage, DNA protection and substrate oxidation.Though evolutionarily related, sequence similarity between families and often withinfamilies is low. To analyse the evolutionary relationships between individual familiesand their functional roles systematically, we turned to 3D structural alignment andphylogenetic methods.ResultsOur phylogenetic network recovers all characterised functional groups of ferritin-likeproteins and suggests evolutionary relationships between them. The evolutionaryhypotheses that are suggested by the phylogeny are tested against availableindependent evidence such as dimerisation geometries, qualitative comparison ofstructures and sequence based partial phylogenies. Generally our hypotheses stand upagainst the evidence, but in a few cases verification have to await further data.ConclusionsTwo large evolutionary groups of ferritin-like proteins are identified from structuralphylogeny: ferritins, bacterioferritins and Dps proteins on one hand, and substrateoxidising proteins, bacterial multicomponent monooxygenases, fatty acid desaturasesand class I ribonucleotide reductase radical generating subunits, on the other. Themethod we present provides a robust way of evolutionarily classifying a functionallydiverse group of distantly related proteins, as well as examining the possible functionsof poorly-characterised proteins.
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| 3. |
- Lundgren, Camilla A. K, et al.
(författare)
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Scavenging of superoxide by a membrane-bound superoxide oxidase
- 2018
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 14, s. 788-793
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Tidskriftsartikel (refereegranskat)abstract
- Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).
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| 4. |
- Covarrubias, Adrian Suarez, et al.
(författare)
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Structural, biochemical and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 381:3, s. 622-633
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Tidskriftsartikel (refereegranskat)abstract
- Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C-gamma in the formation of the alpha beta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth, such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism.
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| 5. |
- Andersson, Charlotta Selina, 1979-
(författare)
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Structural studies of R2 and R2–like proteins with a heterodinuclear Mn/Fe cofactor and enzymes involved in Mycobacterium tuberculosis lipid metabolism
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tuberculosis is a notorious disease responsible for the deaths of 1.4 million people worldwide. A third of the world's population is infected with Mycobacterium tuberculosis, the bacterium causing the disease. The increase of multi drug-resistant strains worsens the situation, and the World Health Organization has declared tuberculosis to be a global emergency. The bacterium envelopes itself with a unique set of very long-chain lipids that play an important role in virulence and drug resistance. Therefore enzymes involved in lipid metabolism are putative drug targets. To allow entry into different metabolic pathways and transmembrane transport, fatty acids have to be activated. This is done primarily by fatty acyl-CoA synthetases (ACSs). We identified an ACS possibly involved in the bacterium’s virulence and solved its structure. Structural interpretation combined with previously reported data gives us insights into the details of its function. This enzyme is known to harbor lipid substrates longer than the enzyme itself, and we now propose how this peripheral membrane protein accommodates its substrates. Some of the most chemically challenging oxidations are performed by dinuclear metalloproteins belonging to the ferritin-like superfamily. We show that the ferritin-like protein, R2lox, from M. tuberculosis contains a new type of heterodinuclear Mn/Fe cofactor. This protein cofactor is capable of performing potent 2-electron oxidations as demonstrated by a novel tyrosine-valine crosslink observed in the protein. Recently a new subclass of ribonucleotide reductase (RNR) R2 proteins, was identified in the intracellular pathogen Chlamydia trachomatis containing the same type of Mn/Fe cofactor mentioned above. The RNR R2 proteins use their metal site to generate a stable radical, essential for the reduction of ribonucleotides to their deoxy forms, the building blocks of DNA. With this work, we were able to characterize the architecture of this metal cofactor.
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| 6. |
- Grāve, Kristīne, 1988-
(författare)
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Structural basis for metalloprotein catalysis : Characterization of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase PgsA1 and Bacillus anthracis ribonucleotide reductase R2
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- About a third of all proteins need to associate with a particular metal ion or metallo-inorganic cofactor to function. This interplay expands the catalytic repertoire of enzymes and reflects the adaption of these catalytic macromolecules to the environments they have evolved in. A large portion of this work focuses on the membrane metalloprotein PgsA1 from the pathogen Mycobacterium tuberculosis and a radical-harboring protein R2 from the pathogen Bacillus anthracis, offering a glimpse into the metalloprotein universe and the catalysis they perform.This thesis is divided into two parts; the first part describes a method for high-throughput M. tuberculosis membrane protein expression screening in Escherichia coli and Mycobacterium smegmatis. This method employs target membrane protein fusions with the folding reporter Green Fluorescent Protein, allowing for fast selection of well-expressing membrane protein targets for further structural and functional characterization. This technique allowed overexpression of M. tuberculosis phosphatidylinositol phosphate synthase PgsA1, leading to its crystallization and the characterization of its high-resolution three-dimensional structure. PgsA1 is a MgII- dependent enzyme, catalyzing a vital step in the biosynthesis of phosphatidylinositol – one of the major phospholipids comprising the complex mycobacterial cell envelope. Therefore, PgsA1 presents an attractive target for the development of new antibiotics against tuberculosis.The second part of this thesis concerns the structural characterization of the B. anthracis class Ib ribonucleotide reductase radical-generating subunit R2 (R2b). R2b contains a dinuclear metallocofactor, which is able to be activated by dioxygen and generates a stable tyrosyl radical; the radical is further used for initiation of nucleotide reduction in the catalytic subunit of ribonucleotide reductase. R2b proteins utilize a di-manganese cofactor in vivo, but can also generate the radical using a di-iron cofactor in vitro, albeit less efficiently. How does R2b achieve correct metallation for efficient catalysis? We show that the B. anthracis R2b protein scaffold is able to select manganese over iron, and furthermore, describe the structural features that govern this metal-specificity. In addition, we describe redox-dependent structural changes in di-iron B. anthracis R2b after reaction with O2, and propose their role in gating solvent access to the metallocofactor and the radical site.
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| 7. |
- Tarry, Michael, et al.
(författare)
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Production of human tetraspanin proteins in Escherichia coli
- 2012
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 82:2, s. 373-379
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Tidskriftsartikel (refereegranskat)abstract
- Tetraspanins are found in multicellular eukaryotes and are generally thought to act as scaffolding proteins, localizing multiple proteins to a specific region of the cell membrane. Activities for tetraspanins have been identified in several fundamental processes such as motility, cell adhesion, proliferation and viral entry. Tetraspanins are also key players in cancer development and progression. However, structural and biochemical information on tetraspanins is decidely limited, due in no small part to the difficulties associated with expressing eukaryotic membrane proteins. In this study, we have used GFP fusions of a library of human tetraspanin proteins to identify growth conditions for expression in Escherichia coli. Three tetraspanin-GFP proteins could be produced at high enough levels to allow subsequent purification, paving the way for future structural and biochemical studies.
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| 8. |
- Martínez-Carranza, Markel, et al.
(författare)
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A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site
- 2020
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 295:46, s. 15576-15587
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1–R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1–R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1–R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.
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| 9. |
- Diamanti, Riccardo, et al.
(författare)
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Comparative structural analysis provides new insights into the function of R2-like ligand-binding oxidase
- 2022
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Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:12, s. 1600-1610
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Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.
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| 10. |
- Grāve, Kristīne, et al.
(författare)
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The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron
- 2020
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 25:4, s. 571-582
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Tidskriftsartikel (refereegranskat)abstract
- Correct protein metallation in the complex mixture of the cell is a prerequisite for metalloprotein function. While some metals, such as Cu, are commonly chaperoned, specificity towards metals earlier in the Irving-Williams series is achieved through other means, the determinants of which are poorly understood. The dimetal carboxylate family of proteins provides an intriguing example, as different proteins, while sharing a common fold and the same 4-carboxylate 2-histidine coordination sphere, are known to require either a Fe/Fe, Mn/Fe or Mn/Mn cofactor for function. We previously showed that the R2lox proteins from this family spontaneously assemble the heterodinuclear Mn/Fe cofactor. Here we show that the class Ib ribonucleotide reductase R2 protein from Bacillus anthracis spontaneously assembles a Mn/Mn cofactor in vitro, under both aerobic and anoxic conditions, when the metal-free protein is subjected to incubation with Mn-II and Fe-II in equal concentrations. This observation provides an example of a protein scaffold intrinsically predisposed to defy the Irving-Williams series and supports the assumption that the Mn/Mn cofactor is the biologically relevant cofactor in vivo. Substitution of a second coordination sphere residue changes the spontaneous metallation of the protein to predominantly form a heterodinuclear Mn/Fe cofactor under aerobic conditions and a Mn/Mn metal center under anoxic conditions. Together, the results describe the intrinsic metal specificity of class Ib RNR and provide insight into control mechanisms for protein metallation.
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