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ABSTRACT Recombinant phenol hydroxylase from the soil yeast Trichosporon cutaneum has been crystallized with PEG 4000 as precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 101.8 A, b = 153.0 A, c = 116.0... more
ABSTRACT Recombinant phenol hydroxylase from the soil yeast Trichosporon cutaneum has been crystallized with PEG 4000 as precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 101.8 A, b = 153.0 A, c = 116.0 A and beta = 114.8 degrees. The crystal asymmetric unit most likely contains two dimers of phenol hydroxylase corresponding to a packing density in the crystals of 2.54 A 3/Da. The self-rotation function is consistent with the packing of two dimers in the asymmetric unit. The observed diffraction pattern extends beyond 2.8 A resolution and the crystals are well suited for structural analysis by X-ray diffraction methods.
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ABSTRACT The thermoresistant gluconate kinase GntK from Escherichia coli, an essential enzyme in gluconate metabolism, has been expressed, purified and crystallized. For crystallization, the hanging-drop vapour-diffusion method was used... more
ABSTRACT The thermoresistant gluconate kinase GntK from Escherichia coli, an essential enzyme in gluconate metabolism, has been expressed, purified and crystallized. For crystallization, the hanging-drop vapour-diffusion method was used with polyethylene glycol (PEG) 6000 and lithium chloride as precipitants. Three crystal forms belonging to the monoclinic space group C2 or the orthorhombic space groups P2(1)2(1)2(1) and P2(1)2(1)2 were obtained. The unit-cell parameters are a = 75.0, b = 79.3, c = 70.2 A, beta = 105.3 degrees (C2), a = 52.0, b = 79.3, c = 89.8 A (P2(1)2(1)2(1)) and a = 70.1, b = 74.1, c = 78.9 A (P2(1)2(1)2). In all three crystal forms, there are two molecules in the asymmetric unit; the different forms occur in the same crystallization drop. The crystals diffract to at least 2.0 A using synchrotron radiation.
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Summary. Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive disorder characterized by mild‐to‐moderate bleeding and reduction in FV and FVIII levels in plasma. F5F8D is caused... more
Summary. Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive disorder characterized by mild‐to‐moderate bleeding and reduction in FV and FVIII levels in plasma. F5F8D is caused by mutations in one of two different genes, LMAN1 and MCFD2, which encode proteins that form a complex involved in the transport of FV and FVIII from the endoplasmic reticulum to the Golgi apparatus. Here, we report the identification of a novel mutation Asp89Asn in the MCFD2 gene in a Tunisian patient. In the encoded protein, this mutation causes substitution of a negatively charged aspartate, involved in several structurally important interactions, to an uncharged asparagine. To elucidate the structural effect of this mutation, we performed circular dichroism (CD) analysis of secondary structure and stability. In addition, CD analysis was performed on two missense mutations found in previously reported F5F8D patients. Our results show that all anal...
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ABSTRACT Crystals of the flavin domain of corn nitrate reductase expressed in Escherichia coli have been obtained at room temperature, using sodium citrate as precipitant. The crystals diffract to at least 2.5 A resolution at a... more
ABSTRACT Crystals of the flavin domain of corn nitrate reductase expressed in Escherichia coli have been obtained at room temperature, using sodium citrate as precipitant. The crystals diffract to at least 2.5 A resolution at a synchrotron radiation source. Precession photographs show that they belong to the rhombohedral space group R3 with unit cell dimensions a = b = 145.4 A, c = 47.5 A, alpha = beta = 90 degrees and gamma = 120 degrees. There is one subunit per asymmetric unit which gives a packing density of 3.2 A3/Da, indicating a high solvent content in these crystals.
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Research Interests: Chemistry, Biological Chemistry, Medicine, Biological Sciences, Phosphatase, and 15 moreAnimals, Male, Prostate, Glycosylation, CHEMICAL SCIENCES, Electrons, Hydrogen Bonding, Rats, Moths, Protein Conformation, Amino Acid Sequence, Molecular Sequence Data, Acid Phosphatase, binding sites, and Medical and Health Sciences
Research Interests: Biochemistry, Biology, Enzyme Inhibitors, Medicine, Biological Sciences, and 15 moreCrystal structure, Computer Simulation, Enzyme, Enzyme Mechanism, CHEMICAL SCIENCES, Active site, Chemical Reaction, Antifungal Agents, Amino Acid Sequence, Ascomycota, Biochemistry and cell biology, Magnaporthe Grisea, Molecular Sequence Data, binding sites, and nadp
Research Interests: Computer Graphics, Chemistry, Crystallography, Molecular Biology, Calcium, and 15 moreCrystallization, Medicine, Crystal structure, Humans, Metals, Hydrogen Bonding, Refinement, Protein Conformation, Amino Acid Sequence, Crystalline Structure, Binding Site, Calcium Ions, Biochemistry and cell biology, Molecular Sequence Data, and binding sites
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ABSTRACT Formyl-CoA transferase from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of formyl-CoA transferase were grown at 293 K using polyethylene glycol 4000... more
ABSTRACT Formyl-CoA transferase from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of formyl-CoA transferase were grown at 293 K using polyethylene glycol 4000 as a precipitant. The diffraction pattern of flash-frozen crystals at 100 K extends to 2.2 A resolution with synchrotron radiation (lambda = 0.933 nm). The crystals are tetragonal and belong to space group I4, with unit-cell parameters a = b = 151.44, c = 99.49 A. The asymmetric unit contains one dimer and the solvent content is 53%. Formyl-CoA transferase was crystallized both as the apoenzyme and as its complex with coenzyme A.
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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has attracted a lot of interest due to its central role in the carbon metabolism of plants and photosynthetic microorganisms (for a review see (1)). The dual function of this... more
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has attracted a lot of interest due to its central role in the carbon metabolism of plants and photosynthetic microorganisms (for a review see (1)). The dual function of this enzyme, catalyzing the primary steps in both photosynthetic carbon dioxide fixation and photorespiration (Figure 1), makes it a challenging target for attempts to improve the efficiency of photosynthesis. Recombinant DNA-techniques provide a promising tool to modify the carboxylase/oxygenase ratio by genetic engineering. However, the application of these techniques requires a detailed knowledge of the catalytic mechanism of the enzyme and the structure of its active site.
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Research Interests: Biochemistry, Chemistry, Carbon, Biology, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Saccharomyces cerevisiae, Humans, Yeast, Glycolysis, Gene, CHEMICAL SCIENCES, Fermentation, Amino Acids, Biosynthesis, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, and Medical and Health Sciences
The amino acid sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum has been fitted to the electron density maps. The resulting protein model has been refined to a nominal resolution of 1.7 A using the... more
The amino acid sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum has been fitted to the electron density maps. The resulting protein model has been refined to a nominal resolution of 1.7 A using the constrained-restrained least-squares refinement program of Sussman and the restrained least-squares refinement program of Hendrickson & Konnert. The crystallographic refinement, based on 76,452 reflections with F greater than sigma (F) in the resolution range 5.5 to 1.7 A resulted in a crystallographic R-factor of 18.0%. The asymmetric unit contains one dimeric ribulose-1,5-biphosphate carboxylase molecule, consisting of 869 amino acid residues and 736 water molecules. The geometry of the refined model is close to ideal, with root-mean-square deviations of 0.018 A in bond lengths and 2.7 degrees in bond angles. Two loop regions, comprising residues 54 to 63 and 324 to 335, and the last ten amino acid residues at the C terminus are disordered in our crystals. The expected trimodal distribution is obtained for the side-chain chi 1-angles with a marked preference for staggered conformation. The hydrogen-bonding pattern in the N-terminal beta-sheet and the parallel sheet in the beta/alpha-barrel is described. A number of hydrogen bonds and salt bridges are involved in domain-domain and subunit-subunit interactions. The subunit-subunit interface in the dimer covers an area of 2800 A2. Considerable deviations from the local 2-fold symmetry are found at both the N terminus (residues 2 to 5) and the C terminus (residues 422 to 457). Furthermore, loop 8 in the beta/alpha-barrel domain has a different conformation in the two subunits. A number of amino acid side-chains have different conformations in the two subunits. Most of these residues are located at the surface of the protein. An analysis of the individual temperature factors indicates a high mobility of the C-terminal region and for some of the loops at the active site. The positions and B-factors for 736 solvent sites have been refined (average B: 45.9 A2). Most of the solvent molecules are bound as clusters to the protein. The active site of the enzyme, especially the environment of the activator Lys191 in the non-activated enzyme is described. Crystallographic refinement at 1.7 A resolution clearly revealed the presence of a cis-proline at the active site. This residue is part of the highly conserved region Lys166-Pro167-Lys168.
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Research Interests: Biochemistry, Chemistry, Biological Chemistry, Medicine, Biological Sciences, and 14 moreSaccharomyces cerevisiae, Sequence alignment, Biological, Yeast, Vegetables, Enzyme, CHEMICAL SCIENCES, Spinach, Amino Acid Sequence, Structure activity Relationship, Flavoprotein, Molecular Sequence Data, binding sites, and Medical and Health Sciences
An electron density map to 2.9 Å (1 Å = 10 -10 m) resolution of Rubisco from Rhodospirillum rubrum has been obtained from crystals of the gene product expressed in Escherichia coli . These crystals are monoclinic, space group P2 1 with... more
An electron density map to 2.9 Å (1 Å = 10 -10 m) resolution of Rubisco from Rhodospirillum rubrum has been obtained from crystals of the gene product expressed in Escherichia coli . These crystals are monoclinic, space group P2 1 with cell dimensions a = 65.5 Å, b = 70.6 Å, c = 104.1 Å and β = 92° and with two subunits per asymmetric unit. Isomorphous phases were obtained from three heavy atom derivatives. The dimeric molecule has the shape of a distorted ellipsoid with approximate dimensions 45 x 70 x 105 Å. The subunit interactions in the dimeric molecule are tight and extensive. Each subunit is divided into two main domains, one of which has extensive α/β structure probably folded into the common eight unit barrel structure. NADPH and pyridoxal phosphate bind at one end of this domain in each subunit. Two different octameric molecules with approximate (422) symmetry have been constructed from the L 2 dimeric molecule from R. rubrum , assuming that the molecular twofold axis of t...