| 1. |
- Andersson, C David, et al.
(författare)
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Discovery of Ligands for ADP-Ribosyltransferases via Docking-Based Virtual Screening
- 2012
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 55:17, s. 7706-7718
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Tidskriftsartikel (refereegranskat)abstract
- The diphtheria toxin-like ADP-ribosyltransferases (ARTDs) are an enzyme family that catalyses the transfer of ADP-ribose units onto substrate proteins, using nicotinamide adenine dinucleotide (NAD(+)) as a co-substrate. They have a documented role in chromatin remodelling and DNA repair; and inhibitors of ARTD1 and 2 (PARP1 and 2) are currently in clinical trials for the treatment of cancer. The detailed function of most other ARTDs is still unknown. Using virtual screening we identified small ligands of ARTD7 (PARP15/BAL3) and ARTD8 (PARP14/BAL2). Thermal-shift assays confirmed that 16 compounds, belonging to eight structural classes, bound to ARTD7/ARTD8. Affinity measurements with isothermal titration calorimetry for two isomers of the most promising hit compound confirmed binding in the low micromolar range to ARTD8. Crystal structures showed anchoring of the hits in the nicotinamide pocket. These results form a starting point in the development of chemical tools for the study of the role and function of ARTD7 and ARTD8.
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| 2. |
- Collins, Ruairi, et al.
(författare)
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Biochemical discrimination between selenium and sulfur 1 : a single residue provides selenium specificity to human selenocysteine lyase
- 2012
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Ingår i: PLoS One. - Stockholm : Karolinska Institutet, Dept of Medical Biochemistry and Biophysics. - 1932-6203.
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Tidskriftsartikel (refereegranskat)abstract
- Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.
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| 3. |
- Dufe, Veronica T, et al.
(författare)
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Cloning, expression, characterisation and three-dimensional structure determination of Caenorhabditis elegans spermidine synthase
- 2005
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 579:27, s. 6037-6043
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Tidskriftsartikel (refereegranskat)abstract
- The polyamine synthesis enzyme spermidine synthase (SPDS) has been cloned from the model nematode Caenorhabditis elegans. Biochemical characterisation of the recombinantly expressed protein revealed a high degree of similarity to other eukaryotic SPDS with the exception of a low affinity towards the substrate decarboxylated S-adenosylmethionine (K-m = 110 mu M) and a less pronounced feedback inhibition by the second reaction product 5 '-methylthioadenosine (IC50 = 430 mu M). The C elegans protein that carries a nematode-specific insertion of 27 amino acids close to its N-terminus was crystallized, leading to the first X-ray structure of a dimeric eukaryotic SPDS. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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| 4. |
- Ekblad, Torun, et al.
(författare)
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Towards small molecule inhibitors of mono-ADP-ribosyltransferases
- 2015
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234 .- 1768-3254. ; 95, s. 546-551
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Tidskriftsartikel (refereegranskat)abstract
- Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50.
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| 5. |
- Hansson, Mattias, et al.
(författare)
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Amino acid residues His183 and Glu264 in Bacillus subtilis ferrochelatase direct and facilitate the insertion of metal ion into protoporphyrin IX
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:1, s. 87-94
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Tidskriftsartikel (refereegranskat)abstract
- Ferrochelatase catalyzes the terminal step in the heme biosynthetic pathway, i.e., the incorporation of Fe(II) into protoporphyrin IX. Various biochemical and biophysical methods have been used to probe the enzyme for metal binding residues and the location of the active site. However, the location of the metal binding site and the path of the metal into the porphyrin are still disputed. Using site-directed mutagenesis on Bacillus subtilis ferrochelatase we demonstrate that exchange of the conserved residues His183 and Glu264 affects the metal affinity of the enzyme. We also present the first X-ray crystal structure of ferrochelatase with iron. Only a single iron was found in the active site, coordinated in a square pyramidal fashion by two amino acid residues, His183 and Glu264, and three water molecules. This iron was not present in the structure of a His183Ala modified ferrochelatase. The results strongly suggest that the insertion of a metal ion into protoporphyrin IX by ferrochelatase occurs from a metal binding site represented by His183 and Glu264.
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| 6. |
- Hansson, Mattias, et al.
(författare)
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Bacterial ferrochelatase turns human: Tyr13 determines the apparent metal specificity of Bacillus subtilis ferrochelatase.
- 2011
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 1432-1327 .- 0949-8257. ; 16:2, s. 235-242
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Tidskriftsartikel (refereegranskat)abstract
- Ferrochelatase catalyzes the insertion of Fe(2+) into protoporphyrin IX. The enzymatic product heme (protoheme IX) is a well-known cofactor in a wide range of proteins. The insertion of metal ions other than Fe(2+) occurs rarely in vivo, but all ferrochelatases that have been studied can insert Zn(2+) at a good rate in vitro. Co(2+), but not Cu(2+), is known to be a good substrate of the mammalian and Saccharomyces cerevisiae ferrochelatases. In contrast, Cu(2+), but not Co(2+), has been found to be a good substrate of bacterial Bacillus subtilis ferrochelatase. It is not known how ferrochelatase discriminates between different metal ion substrates. Structural analysis of B. subtilis ferrochelatase has shown that Tyr13 is an indirect ligand of Fe(2+) and a direct ligand of a copper mesoporphyrin product. A structure-based comparison revealed that Tyr13 aligns with a Met residue in the S. cerevisiae and human ferrochelatases. Tyr13 was changed to Met in the B. subtilis enzyme by site-directed mutagenesis. Enzymatic measurements showed that the modified enzyme inserted Co(2+) at a higher rate than the wild-type B. subtilis ferrochelatase, but it had lost the ability to use Cu(2+) as a substrate. Thus, the B. subtilis Tyr13Met ferrochelatase showed the same metal specificity as that of the ferrochelatases from S. cerevisiae and human.
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| 7. |
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| 8. |
- Karlberg, Tobias, et al.
(författare)
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14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3 beta: ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.
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| 9. |
- Karlberg, Tobias
(författare)
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Iron in Haem Biosynthesis. Structural Studies of Ferrochelatase and Frataxin.
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ferrochelatase catalyses the synthesis of heme by inserting Fe(II) into protoporphyrin IX. The structure of ferrochelatase from yeast has been determined (2.4 Å). The structure is homodimeric with each of the monomer made up of two Rossmann type domains with an active-site cleft between them. Metal binding studies with Co(II) and Cd(II) ions reveal the metal binding residues as being the His-Glu couple, His235 and Glu314. In addition Cd(II) binds to a second site coordinating Glu97, His317 and Glu326.Metal binding studies on the bacterial ferrochelatase from Bacillus subtilis revealed an Fe(II) bound to the same His-Glu couple, His183 and Glu264. The structure was refined to 1.7 Å. Site-directed mutants in the active site were studied and four of them were structurally determined, Tyr13Phe (2.4 Å), Lys87Ala (1.2 Å), His183Cys (2.1 Å) and His183Ala (2.4 Å). In combination with biochemical studies a likely mechanism of metal binding is suggested to involve the His-Glu couple. The porphyrin inhibitor N-MeMP binds with high affinity to ferrochelatase. Cu(II) insertion into N-MeMP was studied by a time series of soaking N-MeMP:ferrochelatase crystals. Cu(II) was seen accumulating into the ring while density for the N-methyl group was seen decreasing. Mass spectrometry studies on porphyrin metallation revealed that ferrochelatase catalyse metal insertion. Cu(II) was more efficiently inserted than Zn(II) which contrasts with studies on the substrate protoporphyrin IX. The most probable reason for the discrepancy is the degree of distortion of the macro-cycle, which therefore could be of importance in metal selectivity. Co-crystal structures of two different inhibitors were determined. N-MeMP crystallised with His183Ala variant of ferrochelatase revealed a different isomer, from the racemic mixture, located in the binding cleft compared to previously determined wild-type structure. The structure was refined to 2.4 Å. His183 is put forward as an important residue controlling binding specificity. The other inihibitor, 2,4-disulphonic deuteroporphyrin IX, has two bulky side-chains and was seen trapped on its way into the binding cleft of the His183Cys ferrochelatase variant (2.1 Å). Frataxin is a mitochondrial protein involved in iron delivery and storage. The crystal structure of trimeric frataxin from yeast was determined to 3.0 Å. The structure has a propeller-like shape with the monomers making up the wings. Each monomer is folded as an alpha/beta sandwich. The same fold was also found in other metallochaperones. In an EM-reconstruction of the 24-meric oligomer the trimers were located at the corners with octahedral symmetry. Eight trimer structures could be docked into the envelope. Frataxin seems to be more dynamic in terms of its bi-functionality and iron storage properties compared to ferritin.
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| 10. |
- Karlberg, Tobias, et al.
(författare)
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Metal Binding to Saccharomyces cerevisiae Ferrochelatase
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:46, s. 13499-13506
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Tidskriftsartikel (refereegranskat)abstract
- Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway. It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX. The crystal structures of ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work. The enzyme is a homodimer, with clear asymmetry between the monomers with regard to the porphyrin binding cleft and the mode of metal binding. The Co(II) and Cd(II) complexes reveal the metal binding site which consists of the invariant amino acids H235, E314, and S275 and solvent molecules. The shortest distance to the metal reveals that amino acid H235 is the primary metal binding residue. A second site with bound Cd(II) was found close to the surface of the molecule, approximately 14 Å from H235, with E97, H317, and E326 participating in metal coordination. It is suggested that this site corresponds to the magnesium binding site in Bacillus subtilis ferrochelatase. The latter site is also located at the surface of the molecule and thought to be involved in initial metal binding and regulation.
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