Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to... more
Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that whe...
Research Interests: Biochemistry, Kinetics, Biology, Enzyme Inhibitors, Medicine, and 15 moreMultidisciplinary, Humans, General, Animals, Enzyme, Purine, Trypanosoma brucei, Phosphoribosyltransferase, Protein Conformation, Trypanocidal Agents, Protozoan Proteins, Species Specificity, Amino Acid Sequence, Recombinant Proteins, and Nucleotide
Research Interests: Engineering, Physics, Chemistry, Biology, Medicine, and 15 moreMultidisciplinary, Crystal structure, Humans, Animals, General medicine, PLoS one, Active site, Agricultural and Biological Sciences, Amino Acid Sequence, Protein Binding, Catalytic Triad, Histidine, Aprotinin, Molecular Sequence Data, and plasmin
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Research Interests: Biophysics, Chemistry, Crystallography, Crystallization, Medicine, and 15 moreGene expression, Macromolecular X-Ray Crystallography, Biological Sciences, Crystal structure, Enzymes, Escherichia coli, Physical sciences, Diffraction, Resolution, Enzyme, CHEMICAL SCIENCES, Biosynthesis, MAD, Molecular Replacement, and C
ABSTRACT Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and... more
ABSTRACT Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) cell entry. The gD ectodomain consists of 316 residues and has no sequence homology to any other proteins of known structure. Two fragments of the HSV-1 gD ectodomain (gD(22-260): residues 22-260 and gD(285): residues 1-285) have been crystallized in two crystal forms. The complex between gD(285) and the ectodomain of HveA, a gD cellular receptor member of the tumor necrosis factor (TNFR) superfamily, has also been crystallized. Moreover, insect-cell-expressed selenomethionine-substituted gD(285) has been purified and crystallized alone and in complex with HveA.
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Research Interests: Biochemistry, Chemistry, Catalysis, Kinetics, Medicine, and 14 moreEnzymes, Mutagenesis, Magnesium, Phosphofructokinase, Stereochemistry, Enzyme, Active site, Site-directed Mutagenesis, Equilibrium Constant, Biochemistry and cell biology, binding sites, Medical biochemistry and metabolomics, nadp, and Transition state
Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the first enzyme in the branched-chain amino acid biosynthesis pathway. Five of the most widely used commercial herbicides (i.e. sulfonylureas, imidazolinones, triazolopyrimidines,... more
Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the first enzyme in the branched-chain amino acid biosynthesis pathway. Five of the most widely used commercial herbicides (i.e. sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates and sulfonylamino-cabonyl-triazolinones) target this enzyme. Here we have determined the first crystal structure of a plantAHAS in the absence of any inhibitor (2.9 Å resolution) and itshows that the herbicide-binding site adopts a folded state even in the absence of an inhibitor. This is unexpected because the equivalent regions for herbicide bindingin uninhibited Saccharomyces cerevisiae AHAS crystal structures are either disordered,or adopt a different fold when the herbicide is not present. In addition, the structure provides anexplanation as to why some herbicides are more potent inhibitorsofArabidopsis thaliana AHAS compared to AHASs from other species (e.g.Saccharomyces cerevisiae). The elucidation of the native structure of pl...
Research Interests: Biochemistry, Molecular Biology, Biology, Cell Biology, Medicine, and 15 moreSaccharomyces cerevisiae, Herbicide, Herbicide Resistance, Herbicides, Enzyme, Active site, Inhibitor, Protein Conformation, Amino Acid Sequence, PLANT PROTEINS, Protein Data Bank, Biochemistry and cell biology, Molecular Structure, binding sites, and Medical biochemistry and metabolomics
Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid biosynthesis, a pathway essential to the life-cycle of plants and micro-organisms. The catalytic subunit has thiamin diphosphate (ThDP) and flavin... more
Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid biosynthesis, a pathway essential to the life-cycle of plants and micro-organisms. The catalytic subunit has thiamin diphosphate (ThDP) and flavin adenine dinucleotide (FAD) as indispensable co-factors. A new, high resolution, 2.0 Å crystal structure of Saccharomyces cerevisiae AHAS reveals that the dimer is asymmetric, with the catalytic centres having distinct structures where FAD is trapped in two different conformations indicative of different redox states. Two molecules of oxygen (O2) are bound on the surface of each active site and a tunnel in the polypeptide appears to passage O2 to the active site independently of the substrate. Thus, O2 appears to play a novel "co-factor" role in this enzyme. We discuss the functional implications of these features of the enzyme that have not previously been described.
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Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions... more
Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the iso...
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Research Interests: Chemistry, Crystallization, Medicine, Macromolecular X-Ray Crystallography, Arabidopsis thaliana, and 15 moreCrystal structure, Stereochemistry, Herbicide, Nitrogen, Arabidopsis, Herbicides, Enzyme, Active site, Sulfonylurea, Triazines, C, Biochemistry and cell biology, binding sites, Sulfonamides, and Medical biochemistry and metabolomics
The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit... more
The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 Å resolution) and with the imidazolinone, imazaquin (IQ; 2.8 Å). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 Å of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 Å from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there...
Research Interests: Biochemistry, Chemistry, Crystallization, Medicine, Multidisciplinary, and 14 moreMacromolecular X-Ray Crystallography, Arabidopsis thaliana, Drug Resistance, Arabidopsis, Herbicides, Enzyme, Active site, Amino Acid Profile, Imidazoles, Quinolines, D structure, X Ray Crystallography, Binding Site, and binding sites
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Research Interests: Biochemistry, Mass Spectrometry, Biology, Enzyme Inhibitors, Medicine, and 15 moreBiological Sciences, Humans, Magnesium, Enzyme, Purine, Plasmodium falciparum, Phosphoribosyltransferase, Metabolic pathway, Recombinant Proteins, Protein Binding, Phosphonic Acids, Analogs, Plasmodium Vivax, coenzymes, and Medical and Health Sciences
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Research Interests: Chemistry, Biology, Crystallization, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Crystal structure, Enzymes, Escherichia coli, Stereochemistry, Enzyme, CHEMICAL SCIENCES, Active site, Amino Acid Sequence, C, Thiamine, Klebsiella pneumoniae, Molecular Sequence Data, binding sites, and Medical and Health Sciences
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Research Interests: Biochemistry, Chemistry, Organic Chemistry, Medicinal Chemistry, Enzyme Inhibitors, and 15 moreMedicine, Organic Synthesis, Phosphorus Chemistry, Osteoporosis, Humans, Phosphatase, PAP, Glycoproteins, Enzyme, Hydrolysis, C, Bone Resorption, Acid Phosphatase, phosphonate, and Pharmacology and pharmaceutical sciences
The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect... more
The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect of metal ion substitutions and site-directed mutations on the catalytic properties of OpdA are investigated. The study shows the importance of both the metal ion composition and a hydrogen-bond network that connects the metal ion centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the mechanism and substrate specificity of this enzyme. For the Co(II) derivative of OpdA two protonation equilibria (pKa1 ~5; pKa2 ~10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II) derivatives only have one relevant protonation equilibrium (pKa ~4–5), and the μOH is the proposed nucleophile. The observed mechanistic flexibility may...
Research Interests: Chemistry, Catalysis, Kinetics, Biology, Medicine, and 15 moreBiocatalysis, Biological Sciences, Crystal structure, Heavy metals, Pesticides, Biochemical, Organophosphorus Compounds, Enzyme, Metalloproteins, CHEMICAL SCIENCES, Hydrogen Bonding, Active site, Arginine, Hydroxide, and Medical and Health Sciences
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Research Interests: Biophysics, Crystallography, Biology, Medicine, Macromolecular X-Ray Crystallography, and 15 moreBiological Sciences, Enzymes, Escherichia coli, Physical sciences, Temperature, Expression, Plasmids, Enzyme, CHEMICAL SCIENCES, Purification, Biochemistry and molecular biology, C, Klebsiella pneumoniae, reconstitution, and Valine
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Acetohydroxyacid synthase (AHAS) is the target for more than 50 commercial herbicides; first applied to crops in the 1980s. Since then, 197 site-of-action resistance isolates have been identified in weeds, with mutations at P197 and W574... more
Acetohydroxyacid synthase (AHAS) is the target for more than 50 commercial herbicides; first applied to crops in the 1980s. Since then, 197 site-of-action resistance isolates have been identified in weeds, with mutations at P197 and W574 the most prevalent. Consequently, AHAS is at risk of not being a useful target for crop protection. To develop new herbicides, a functional understanding to explain the effect these mutations have on activity is required. Here, we show that these mutations can have two effects (i) to reduce binding affinity of the herbicides and (ii) to abolish time-dependent accumulative inhibition, critical to the exceptional effectiveness of this class of herbicide. In the two mutants, conformational changes occur resulting in a loss of accumulative inhibition by most herbicides. However, bispyribac, a bulky herbicide is able to counteract the detrimental effects of these mutations, explaining why no site-of-action resistance has yet been reported for this herbic...
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Research Interests: Catalysis, Kinetics, Bone Metabolism, Macromolecular X-Ray Crystallography, Molecular Mechanics, and 15 moreCatalytic Mechanism, Crystal structure, Fluorides, Glycoproteins, Phaseolus, Enzyme, Hydrogen Bond, Active site, Metal ion, Protein Conformation, Reaction Mechanism, PLANT PROTEINS, Ligands, Bone Resorption, and Sulfates
The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a... more
The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a "Bence-Jones protein" from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 A resolution and refined to R and R(free) factors of 0.22 and 0.25, respectively. This "amyloidogenic" protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a = 48.28, b = 83.32, c = 112.59 A as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL).
Research Interests: Biophysics, Crystallography, Protein Folding, Macromolecular X-Ray Crystallography, Biological Sciences, and 16 moreIn Vitro, Humans, Physical sciences, Deposition, Solubility, CHEMICAL SCIENCES, Protein Secondary Structure Prediction, X ray diffraction, Amino Acid Sequence, Fibrillogenesis, Protein Quaternary Structure, Light chain, Immunoglobulin, Static Electricity, Amyloidosis, and Dimerization
Research Interests: Genetics, Phylogenetics, Medicinal Plants, Arabidopsis thaliana, Mammals, and 22 morePhylogeny, Sequence alignment, Ph, Animals, Plants, Gene, Soybean, Glycoproteins, Sequence, Arabidopsis, Vegetables, Enzyme, Molecular cloning, Sweet Potato, Secondary Structure, Active site, Soybeans, Protein Secondary Structure Prediction, Fabaceae, Metal ion, Amino Acid Sequence, and Molecular Sequence Data
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Research Interests: Kinetics, Enzyme Inhibitors, Crystal structure, Humans, Computer Simulation, and 17 moreMice, Animals, Glycoproteins, Phaseolus, Camote Ipomoea Batatas, Enzyme, Phosphotyrosine, Swine, Substrate Specificity, Species Specificity, Protein Binding, Hydrolysis, Hydrogen-Ion Concentration, Kinetic Parameter, Bone Resorption, Phosphopeptides, and Biochemistry and cell biology
Research Interests: Molecular Biology, Enzyme Inhibitors, Macromolecular X-Ray Crystallography, Crystal structure, Magnesium, and 11 moreMutation, Herbicides, Active site, Protein Conformation, Yeasts, Imidazoles, Amino Acid Sequence, Binding Site, Biochemistry and cell biology, Dimerization, and Molecular Sequence Data
Research Interests: Structure, Protein Folding, Macromolecular X-Ray Crystallography, Biological Sciences, Crystal structure, and 12 moreProtein structure, Motion, Structural Change, Hydrogen Bond, CHEMICAL SCIENCES, Active site, Thioredoxin, Protein Conformation, Hydrogen-Ion Concentration, Oxidation-Reduction, Conformational Change, and Disulfide bond
Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the... more
Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro-N-(4′-methyl-pyrimidin−2′-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure–activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.
Research Interests: Enzyme Inhibitors, Arabidopsis thaliana, Saccharomyces cerevisiae, Crystal structure, Computer Simulation, and 13 moreDrug Design, Quantitative Structure Activity Relationship, Arabidopsis, Herbicides, Enzyme, Rational drug design, Three Dimensional, THEORETICAL AND COMPUTATIONAL CHEMISTRY, Molecular Conformation, Protein Conformation, Structure activity Relationship, Recombinant Proteins, and Computer
DsbA is a protein-folding catalyst from the periplasm of Escherichia coli that interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the... more
DsbA is a protein-folding catalyst from the periplasm of Escherichia coli that interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the interaction between DsbA and unfolded substrate is not known. Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 Å, and present a proposal for its interaction with peptide.The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction. A hinge point for domain motion is identified—the type IV β-turn Phe 63-Met 64-Gly 65-Gly 66, which connects the two domains.Three unique features on the active site surface of the DsbA molecule—a groove, hydrophobic pocket, and hydrophobic patch—form an extensive uncharged surface surrounding the active-site disulfide. Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues. Furthermore, the residues immediately surrounding the active-site disulfide are uncharged in all nine DsbA proteins.A model for DsbA-peptide interaction has been derived from the structure of a human thioredoxin:peptide complex. This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin. The active-site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.
Research Interests: Protein Folding, Protein Science, Macromolecular X-Ray Crystallography, Protein crystallography, Identification, and 11 moreEscherichia coli, Sequence, Active site, Thioredoxin, Protein Conformation, Amino Acid Sequence, Protein Binding, Isomerases, Oxidation-Reduction, Biochemistry and cell biology, and Molecular Sequence Data
The crystal structure of human ubiquitin has been solved by x-ray diffraction methods and refined by standard procedures to a conventional crystallographic R factor of 0.176 at 1.8-A resolution (Vijay-Kumar, S., Bugg, C.E., and Cook, W.J.... more
The crystal structure of human ubiquitin has been solved by x-ray diffraction methods and refined by standard procedures to a conventional crystallographic R factor of 0.176 at 1.8-A resolution (Vijay-Kumar, S., Bugg, C.E., and Cook, W.J. (1987) J. Mol. Biol. 194, 525-538). Crystals of yeast and oat ubiquitin have been grown using human ubiquitin crystals as seeds. Diffraction data for yeast and oat ubiquitin have been collected to a resolution of 1.9 and 1.8 A, respectively. Difference Fourier electron-density maps reveal that the structures of yeast and oat ubiquitin are quite similar to human ubiquitin. All the amino acid changes are clustered in two small patches on one surface of the molecule. This surface is probably not involved in conjugation with proteins destined for ATP-dependent proteolysis.
Research Interests: Water, Molecular Biology, Protein Folding, Crystallization, Macromolecular X-Ray Crystallography, and 30 moreBiological Sciences, Molecular Recognition, Molecular, Glutamate, Humans, Sequence alignment, Computer Simulation, Mice, Animals, Biological, Iron, Horses, Cereals, Solubility, Metals, CHEMICAL SCIENCES, Electrons, Hydrogen Bonding, High Resolution, Protein Secondary Structure Prediction, Ceruloplasmin, X ray diffraction, Protein Conformation, Yeasts, Amino Acid Sequence, Ions, Light chain, Biochemistry and cell biology, Interferon gamma, and Molecular Sequence Data
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Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I)... more
Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I) found in fungi and most bacteria, and a long form (Class II) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 Å resolution, the first three-dimensional structure of any bacterial Class II KARI. The enzyme consists of two domains, one with mixed α/β structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly α-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.
Research Interests: Molecular Evolution, Protein Science, Calcium, Protein, Macromolecular X-Ray Crystallography, and 10 moreCrystal structure, Sequence alignment, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas Aeruginosa, Amino Acid Sequence, Protein Quaternary Structure, Biochemistry and cell biology, Dimerization, and Molecular Sequence Data
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Research Interests: Biophysics, Biological Sciences, Crystal structure, Collagen, Plant, and 15 morePhysical sciences, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Enzyme, Cleavage, MMP, Matrix Metalloproteinases, Rhizome, Cysteine endopeptidases, Ginger, Amino Acid Sequence, SDS PAGE, Hydrolysis, Biochemistry and cell biology, Type I Collagen, and Molecular Sequence Data
Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Å resolution) and for the enzyme in complex with the reaction products, inosine 5′-monophosphate (IMP) and guanosine... more
Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Å resolution) and for the enzyme in complex with the reaction products, inosine 5′-monophosphate (IMP) and guanosine 5′-monophosphate (GMP) (2.8 Å resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Cα atoms of 1.0 Å. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg2+ in vivo. In the free structure, a Mg2+ is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.
Research Interests: Enzymology, Kinetics, Protein Science, Macromolecular X-Ray Crystallography, Crystal structure, and 11 moreHumans, Sequence alignment, Escherichia coli, Animals, Purine salvage, Molecular cloning, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Amino Acid Sequence, Protein Quaternary Structure, Biochemistry and cell biology, and Molecular Sequence Data
Research Interests: Organic Chemistry, Molecular Biology, Structure, Protein Folding, Catalysis, and 24 moreMacromolecular X-Ray Crystallography, Biological Sciences, Molecular, Crystal structure, Humans, Animals, Drug Design, Expression, Protein structure, Glycoproteins, TRAP, Enzyme, CHEMICAL SCIENCES, Pp, Active site, Rats, Swine, Metal ion, Protein Conformation, Amino Acid Sequence, Chemistry Biology, Bone Resorption, Biochemistry and cell biology, and Molecular Sequence Data
DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and... more
DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys30-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH.His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of -5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein.Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys30. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.