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ABSTRACT: The key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) has been shown to be the target enzyme of fosmidomycin, an antimalarial, antibacterial and herbicidal compound. Here we report the crystal structure of selenomethionine-labelled Escherichia coli DXR in a ternary complex with NADPH and fosmidomycin at 2.2 A resolution. The structure reveals a considerable conformational rearrangement upon fosmidomycin binding and provides insights into the slow, tight binding inhibition mode of the inhibitor. Although the inhibitor displays an unusual non-metal mediated mode of inhibition, which is an artefact most likely due to the low metal affinity of DXR at the pH used for crystallization, the structural data add valuable information for the rational design of novel DXR inhibitors. Using this structure together with the published structural data and the 1.9 A crystal structure of DXR in a ternary complex with NADPH and the substrate 1-deoxy-D-xylulose 5-phosphate, a model for the physiologically relevant tight-binding mode of inhibition is proposed. The structure of the substrate complex must be interpreted with caution due to the presence of a second diastereomer in the active site.
Journal of Molecular Biology 02/2005; 345(1):115-27. · 4.00 Impact Factor
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Alice Douangamath,
Glenn E. Dale,
Allan D'Arcy,
Michael Almstetter,
Robert Eckl,
Annabelle Frutos-Hoener,
Bernd Henkel,
Katrin Illgen,
Sven Nerdinger,
Henk Schulz,
Aengus Mac Sweeney,
Michael Thormann,
Andreas Treml,
Sabine Pierau,
Sjoerd Wadman,
Christian Oefner
12/2004;
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Alice Douangamath,
Glenn E Dale,
Allan D'Arcy,
Michael Almstetter,
Robert Eckl,
Annabelle Frutos-Hoener,
Bernd Henkel,
Katrin Illgen,
Sven Nerdinger,
Henk Schulz,
Aengus Mac Sweeney,
Aengus MacSweeney,
Michael Thormann,
Andreas Treml,
Sabine Pierau,
Sjoerd Wadman,
Christian Oefner
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ABSTRACT: High-resolution crystal structures of Staphylococcus aureus methionine aminopeptidase I in complex with various keto heterocycles and aminoketones were determined, and the intermolecular ligand interactions with the enzyme are reported. The compounds are effective inhibitors of the S. aureus enzyme because of the formation of an uncleavable tetrahedral intermediate upon binding. The electron densities unequivocally show the enzyme-catalyzed transition-state analogue mimicking that for amide bond hydrolysis of substrates.
Journal of Medicinal Chemistry 04/2004; 47(6):1325-8. · 5.25 Impact Factor
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Christian Oefner, Alice Douangamath,
Allan D'Arcy,
Sascha Häfeli,
Daniel Mareque,
Aengus Mac Sweeney,
Juan Padilla,
Sabine Pierau,
Henk Schulz,
Michael Thormann,
Sjoerd Wadman,
Glenn E Dale
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ABSTRACT: Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.
Journal of Molecular Biology 10/2003; 332(1):13-21. · 4.00 Impact Factor
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Alice Douangamath,
Fabian V Filipp,
André T J Klein,
Phil Barnett,
Peijian Zou,
Tineke Voorn-Brouwer,
M Cristina Vega,
Olga M Mayans,
Michael Sattler,
Ben Distel,
Matthias Wilmanns
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ABSTRACT: While the function of most small signaling domains is confined to binary ligand interactions, the peroxisomal Pex13p SH3 domain has the unique capacity of binding to two different ligands, Pex5p and Pex14p. We have used this domain as a model to decipher its structurally independent ligand binding sites. By the combined use of X-ray crystallography, NMR spectroscopy, and circular dichroism, we show that the two ligands bind in unrelated conformations to patches located at opposite surfaces of this SH3 domain. Mutations in the Pex13p SH3 domain that abolish interactions within the Pex13p-Pex5p interface specifically impair PTS1-dependent protein import into yeast peroxisomes.
Molecular Cell 12/2002; 10(5):1007-17. · 14.18 Impact Factor
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ABSTRACT: Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-A resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.
Journal of Biological Chemistry 03/2002; 277(7):5290-8. · 4.77 Impact Factor
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ABSTRACT: Since reactive ammonia is not available under physiological conditions, glutamine is used as a source for the incorporation of nitrogen in a number of metabolic pathway intermediates. The heterodimeric ImGP synthase that links histidine and purine biosynthesis belongs to the family of glutamine amidotransferases in which the glutaminase activity is coupled with a subsequent synthase activity specific for each member of the enzyme family. Its X-ray structure from the hyperthermophile Thermotoga maritima shows that the glutaminase subunit is associated with the N-terminal face of the (beta alpha)(8) barrel cyclase subunit. The complex reveals a putative tunnel for the transfer of ammonia over a distance of 25 A. Although ammonia tunneling has been reported for glutamine amidotransferases, the ImGP synthase has evolved a novel mechanism, which extends the known functional properties of the versatile (beta alpha)(8) barrel fold.
Structure 03/2002; 10(2):185-93. · 6.35 Impact Factor
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[show abstract]
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ABSTRACT: Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the
Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction
projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for
their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target
peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain
solved at 1.3-Å resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon
at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged
side chains may be the key determinant for the preference for non-classical ligands.
Journal of Biological Chemistry 02/2002; 277(7):5290-5298. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: The key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) has been shown to be the target enzyme of fosmidomycin, an antimalarial, antibacterial and herbicidal compound. Here we report the crystal structure of selenomethionine-labelled Escherichia coli DXR in a ternary complex with NADPH and fosmidomycin at 2.2 Å resolution. The structure reveals a considerable conformational rearrangement upon fosmidomycin binding and provides insights into the slow, tight binding inhibition mode of the inhibitor. Although the inhibitor displays an unusual non-metal mediated mode of inhibition, which is an artefact most likely due to the low metal affinity of DXR at the pH used for crystallization, the structural data add valuable information for the rational design of novel DXR inhibitors. Using this structure together with the published structural data and the 1.9 Å crystal structure of DXR in a ternary complex with NADPH and the substrate 1-deoxy-d-xylulose 5-phosphate, a model for the physiologically relevant tight-binding mode of inhibition is proposed. The structure of the substrate complex must be interpreted with caution due to the presence of a second diastereomer in the active site.
Journal of Molecular Biology.
