| 1. |
- Andaloussi, Mounir, et al.
(författare)
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Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase
- 2011
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 54:14, s. 4964-4976
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Tidskriftsartikel (refereegranskat)abstract
- The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.
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| 2. |
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| 3. |
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| 4. |
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| 5. |
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| 6. |
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| 7. |
- Bergfors, Terese, et al.
(författare)
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Screening cells for crystals : a synergistic approach
- 2020
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Ingår i: Journal of applied crystallography. - : International Union of Crystallography (IUCr). - 0021-8898 .- 1600-5767. ; 53, s. 1414-1415
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 8. |
- Bergfors, Terese
(författare)
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Seeds to crystals.
- 2003
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Ingår i: J Struct Biol. - 1047-8477. ; 142:1, s. 66-76
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Tidskriftsartikel (refereegranskat)
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| 9. |
- Bergfors, Terese
(författare)
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Succeeding with seeding : some practical advice
- 2007
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Ingår i: Evolving Methods for Macromolecular Crystallography. - Dordrecht : Springer Netherlands. - 9781402063145 ; , s. 1-10
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Konferensbidrag (refereegranskat)abstract
- Seeding is a powerful and versatile method for optimizing crystal growth conditions. This article discusses, from a practical point of view, what seeding is, the selection and transfer of seeds, and into what conditions they should be transferred. The most common causes of failures in seeding experiments are also analyzed.
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| 10. |
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| 11. |
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| 12. |
- Björkelid, Christofer, et al.
(författare)
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Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:25, s. 18260-18270
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacterium tuberculosis, the bacterial causative agent oftuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemicalcharacterizations of two new classes of compounds that inhibitpantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenateand phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for anypantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.
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| 13. |
- Björkelid, Christofer, et al.
(författare)
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Structural and functional studies of mycobacterial IspD enzymes
- 2011
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 67, s. 403-414
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Tidskriftsartikel (refereegranskat)abstract
- A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.
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| 14. |
- Björkelid, Christofer, et al.
(författare)
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Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
- 2012
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68, s. 134-143
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Tidskriftsartikel (refereegranskat)abstract
- A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.
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| 15. |
- Chofor, Rene, et al.
(författare)
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Synthesis and Bioactivity of beta-Substituted Fosmidomycin Analogues Targeting 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase
- 2015
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 58:7, s. 2988-3001
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Tidskriftsartikel (refereegranskat)abstract
- Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the beta-position of the hydroxamate analogue of 2. While direct addition of a beta-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the longer compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamates methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.
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| 16. |
- 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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| 17. |
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| 18. |
- D'Arcy, Allan, et al.
(författare)
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Microseed matrix screening for optimization in protein crystallization : what have we learned?
- 2014
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Ingår i: Acta Crystallographica Section F. - 2053-230X. ; 70, s. 1117-1126
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Tidskriftsartikel (refereegranskat)abstract
- Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time-and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.
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| 19. |
- Ericsson, Daniel J., et al.
(författare)
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X-Ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 376:1, s. 109-119
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Tidskriftsartikel (refereegranskat)abstract
- In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-Å resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic α/β hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation
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| 20. |
- Fullam, Elizabeth, et al.
(författare)
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Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme
- 2012
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Ingår i: Open Biology. - : The Royal Society. - 2046-2441. ; 2, s. 110026-
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Tidskriftsartikel (refereegranskat)abstract
- The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 angstrom resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.
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| 21. |
- Ge, Xueliang, et al.
(författare)
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Inhibition of translation termination by small molecules targeting ribosomal release factors
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.
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| 22. |
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| 23. |
- Jakobsson, Emma, et al.
(författare)
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The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1.
- 2003
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Ingår i: Biochim Biophys Acta. - 0006-3002. ; 1649:1, s. 40-50
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Tidskriftsartikel (refereegranskat)abstract
- We describe the 1.6 A crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease.The crystal structure reveals that EgFABP1 has the expected 10-stranded beta-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1.The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R em leader R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.
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| 24. |
- Jansson, Anna, 1979-, et al.
(författare)
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DXR Inhibition by Potent Mono- and Disubstituted Fosmidomycin Analogues
- 2013
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 56:15, s. 6190-6199
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Tidskriftsartikel (refereegranskat)abstract
- The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway producing the universally essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the cramped substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM.
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| 25. |
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| 26. |
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| 27. |
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| 28. |
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| 29. |
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| 30. |
- Lu, Lu, 1984-, et al.
(författare)
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MEPicides : α,β-Unsaturated Fosmidomycin N-acyl Analogsas inhibitors that selectively target DXR from Plasmodium falciparum, the deadliest causative parasite of human Malaria
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Fosmidomycin and FR-9000098 have been confirmed to show parasiticidal activity against Plasmodium falciparum, targeting DXR involved in the MEP pathway. We designed a construct of PfDXR that has successfully been overexpressed in E. coli BL21(DE3) C43, and purified by IMAC and SEC, with the final yield of 1.2 mg/ 8 L culture. PfDXR was concentrated to 20 mg/ml, and co-crystallized with previously tested inhibitors in the FR-9000098 scaffold in the presence of Mn2+. Three FR-9000098 analogues with double-bonded Ca-Cband/or a phenyl ring with various lengths to N1, showed inhibitory activities with IC50s roughly 50 nM. Three crystals were in triclinic P1space group, with similar dimensions in the unit cell (51Å, 56Å, 86Å, 103°, 103°, 101°). All four complex structures have been crystallographically determined at resolutions in the range 1.86 Å, 2.45 Å, 2.13Å, 2.05 Å. Given the high similarity in structures, the initial phases were determined by rigid body refinement with search model PfDXR-FN3 complex, followed by restrained refinement in refmac5. Subsequently, the ligands and surrounding amino acid residues were manually rebuilt with theqdstools in O. the Ca-Cbbonds of the three ligands were altered from a single to double bond based on the structure of FR9000098. In addition, two ligands were extended at the Cdwith a phenyl group, and with the benzyl group connected by two carbons. N-terminal NADPH binding domains from four complexes undergo minor rigid body movement, and more details of conformational changes in the flap region are discussed.
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| 31. |
- Majumdar, Soneya, et al.
(författare)
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Crystal Structures of Ancestral Orthologues Reveal the Molecular Basis of Thermostability in Thermophilic EF-Tus
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The molecular basis of protein thermostability is diverse and unclear. To better understand it, we solved high-resolution crystal structures of four 0.5 – 3.5 billion year old ancestral bacterial Elongation Factor-Tus (EF-Tu). Structural comparison revealed two key interactions, unique for the thermophilic EF-Tus; i) a hydrogen bond between a G-domain tyrosine and the α- phosphate of the guanine nucleotide that stabilizes GTP/GDP; and ii) an inter-domain salt-bridge, tethering the Domains II and III via an arginine and a glutamic acid, respectively. We could reverse the thermostability profiles of the thermophilic and mesophilic EF-Tus by adding or removing these interactions, which were validated with aggregation, biophysical, and functional assays. Further, molecular dynamics simulations demonstrated that these interactions contribute to thermostability via the Mg2+ in the nucleotide binding site. Furthermore, the inter-domain interaction restricts the transition between the open and the closed conformations of the EF-Tus, thereby regulating their thermoactivity.
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| 32. |
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| 33. |
- Sooriyaarachchi, Sanjeewani, et al.
(författare)
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Targeting an Aromatic Hotspot in Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase with -Arylpropyl Analogues of Fosmidomycin
- 2016
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Ingår i: ChemMedChem. - : Wiley. - 1860-7179 .- 1860-7187. ; 11:18, s. 2024-2036
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Tidskriftsartikel (refereegranskat)abstract
- Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the -position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P.falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.
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| 34. |
- Stojanoff, Vivian, et al.
(författare)
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From screen to structure with a harvestable microfluidic device
- 2011
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 67, s. 971-975
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Tidskriftsartikel (refereegranskat)abstract
- Advances in automation have facilitated the widespread adoption of high-throughput vapour-diffusion methods for initial crystallization screening. However, for many proteins, screening thousands of crystallization conditions fails to yield crystals of sufficient quality for structural characterization. Here, the rates of crystal identification for thaumatin, catalase and myoglobin using microfluidic Crystal Former devices and sitting-drop vapour-diffusion plates are compared. It is shown that the Crystal Former results in a greater number of identified initial crystallization conditions compared with vapour diffusion. Furthermore, crystals of thaumatin and lysozyme obtained in the Crystal Former were used directly for structure determination both in situ and upon harvesting and cryocooling. On the basis of these results, a crystallization strategy is proposed that uses multiple methods with distinct kinetic trajectories through the protein phase diagram to increase the output of crystallization pipelines.
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| 35. |
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| 36. |
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| 37. |
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| 38. |
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| 39. |
- Ubhayasekera, Wimal, et al.
(författare)
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Crystal structures of a family 19 chitinase from Brassica juncea show flexibility of binding cleft loops
- 2007
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:14, s. 3695-3703
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Tidskriftsartikel (refereegranskat)abstract
- Brassica juncea chitinase is an endo-acting, pathogenesis-related protein that is classified into glycoside hydrolase family 19, with highest homology (50–60%) in its catalytic domain to class I plant chitinases. Here we report X-ray structures of the chitinase catalytic domain from wild-type (apo, as well as with chloride ions bound) and a Glu234Ala mutant enzyme, solved by molecular replacement and refined at 1.53, 1.8 and 1.7 Å resolution, respectively. Confirming our earlier mutagenesis studies, the active-site residues are identified as Glu212 and Glu234. Glu212 is believed to be the catalytic acid in the reaction, whereas Glu234 is thought to have a dual role, both activating a water molecule in its attack on the anomeric carbon, and stabilizing the charged intermediate. The molecules in the various structures differ significantly in the conformation of a number of loops that border the active-site cleft. The differences suggest an opening and closing of the enzyme during the catalytic cycle. Chitin is expected to dock first near Glu212, which will protonate it. Conformational changes then bring Glu234 closer, allowing it to assist in the following steps. These observations provide important insights into catalysis in family 19 chitinases.
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