ABSTRACT
A new monoclinic solvate containing two molecules of the thiopeptide antibiotic thiostrepton in the asymmetric unit has been crystallized in space group P2(1). Single-crystal diffraction data to a resolution of 0.64â Å were collected at the SLS synchrotron, allowing structure solution by direct methods and resolution of the disorder present. Valence electron density can be observed in the Fourier residual density from refinement with the independent-atom model, which is a prerequisite for successful application of more sophisticated aspherical-atom scattering factors such as the invariom model when aiming to improve the structural model. Invariom refinement improves quality indicators such as R1(F) for thiostrepton, as previously demonstrated for small molecules. The nonspherical electron-density model also allows the direct derivation of a dipole moment and an electrostatic potential for the whole molecule, which is discussed in the context of antibiotic activity and molecular recognition.
Subject(s)
Models, Molecular , Thiostrepton/chemistry , Anti-Bacterial Agents/chemistry , Crystallography, X-Ray , Electrons , Hydrogen Bonding , Static ElectricityABSTRACT
CONTEXT: The T(201)M variant (rs28757184) within exon 5 of the human aromatase gene CYP19A1, present in up to 20% of some populations, has been reported to reduce prostate cancer progression. OBJECTIVE: We hypothesized that the T(201)M variant would alter the structure of the enzyme and thus would also affect function compared to wild-type human aromatase. DESIGN: HEK293 cells were transiently transfected with CYP19A1 wild-type or T(201)M variant gene transcripts made by site-directed mutagenesis and enzyme activity measured using tritiated androstenedione as the substrate. The effects of differing concentrations of substrate and product (E1 and E2) and four aromatase inhibitors were assessed. RESULTS: At all substrate concentrations tested, the T(201)M variant showed substantially increased activity compared to the wild-type (Vmax: variant, 738 +/- 36 pmol/h . mg; wild-type, 189 +/- 17 pmol/h . mg, P < 0.0001; Km: variant, 64.4 +/- 19.3 nm; wild-type, 46.6 +/- 9.1 nm, P = 0.04). Kinetic analysis showed evidence of substrate inhibition for the wild-type, but no product inhibition was demonstrated for either transcript. Formestane, chrysin, and letrozole had no differential inhibitory effect on the two transcripts, but aminoglutethimide inhibition was substantially reduced in the variant compared to wild-type (IC(50): wild-type, 1.3 +/- 0.2 nm; variant, 45 +/- 14.2 nm, P = 0.002; and Ki: wild-type, 0.7 +/- 0.2 nm; variant, 29.6 +/- 9.7 nm, P = 0.0001). CONCLUSIONS: In addition to loss of function mutations previously described, a new naturally occurring relatively common alteration of enzyme structure at T(201)M increases enzyme activity and reduces the inhibitory effect of aminoglutethimide. These findings identify the T(201)M site, distant from the substrate-binding site and not previously considered to play a role in enzyme activity, as a functionally important area of the enzyme that may play a role in the propensity to disease. Common to other cytochrome P450 enzymes, wild-type aromatase demonstrates substrate but not product inhibition.
Subject(s)
Aromatase Inhibitors/pharmacology , Aromatase/genetics , Aminoglutethimide/pharmacology , Androstenedione/metabolism , Aromatase/chemistry , Aromatase/metabolism , Cell Line , Humans , Kinetics , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
PPR (pentatricopeptide repeat) genes form a large family particularly prevalent in higher plants and targeted to organelles. They are involved in many post-transcriptional processes such as splicing, editing, processing and translation. Current data suggest that PPR proteins are involved in targeting effectors to the correct sites on the correct transcripts but the molecular mechanisms for RNA binding and effector recruitment by PPR proteins are not understood yet.
Subject(s)
Organelles/genetics , Plant Proteins/genetics , RNA Processing, Post-Transcriptional/genetics , Animals , Humans , Organelles/metabolism , Plant Development , Plant Proteins/metabolism , Plants/genetics , Plants/metabolism , RNA, Plant/genetics , RNA, Plant/metabolismABSTRACT
The function, structure and mechanism of two Escherichia coli enzymes involved in the non-mevalonate route of isoprenoid biosynthesis, 2C-methyl-D-erythritol 4-phosphate cytidylyltransferase and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, are reviewed. Comparisons of each with enzymes from microbial pathogens highlight important conservation of sequence suggestive of similarities in secondary structure, subunit folds, quaternary structure and active sites. Since both enzymes are validated drug targets, the models provide templates for structure-based design of anti-microbial agents targeting a number of serious human diseases.
Subject(s)
Escherichia coli/enzymology , Insect Proteins/chemistry , Insect Proteins/metabolism , Protein Prenylation/physiology , Amino Acid Sequence , Binding Sites , Mevalonic Acid/metabolism , Models, Molecular , Protein Conformation , Sequence AlignmentABSTRACT
The large quantity of genomic, biochemical and metabolic data on microbial pathogens provides information that helps us to select biological problems of interest and to identify targets, metabolic pathways or constituent enzymes, for therapeutic intervention. One area of potential use in developing novel anti-parasitic agents concerns the regulation of oxidative stress, and we have targeted the trypanothione peroxidase pathway in this respect. In order to characterize this pathway, we have determined crystal structures for each of its components, and are now studying enzyme-ligand complexes of the first enzyme, trypanothione reductase. Also with regard to trypanosomatids, a question that arose was: why do anti-folates not provide useful therapies? The enzyme pteridine reductase has been shown to contribute to anti-folate drug resistance, and we have determined the enzyme structure and mechanism to understand this aspect of drug resistance.
Subject(s)
Folic Acid Antagonists/pharmacology , Oxidative Stress , Trypanosomatina/drug effects , Animals , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Models, Molecular , Oxidation-Reduction , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Peroxidases/antagonists & inhibitors , Peroxidases/chemistry , Peroxidases/metabolism , Protein Conformation , Trypanosomatina/enzymologyABSTRACT
Diphosphocytidyl-methylerythritol (DPCME) synthetase is involved in the mevalonate-independent pathway of isoprenoid biosynthesis, where it catalyses the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol 4-phosphate and CTP. The Escherichia coli enzyme has been cloned, expressed in high yield, purified and crystallized. Elongated tetragonal prismatic crystals grown by the hanging-drop vapour-diffusion method using polyethylene glycol (PEG) 4000 as the precipitant belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 73.60, c = 175.56 A. Diffraction data have been recorded to 2.4 A resolution using synchrotron radiation.
Subject(s)
Escherichia coli/enzymology , Nucleotidyltransferases/chemistry , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Nucleotidyltransferases/biosynthesis , Nucleotidyltransferases/genetics , Protein Conformation , Recombinant Proteins/chemistryABSTRACT
The 2.15-A structure of Hjc, a Holliday junction-resolving enzyme from the archaeon Sulfolobus solfataricus, reveals extensive structural homology with a superfamily of nucleases that includes type II restriction enzymes. Hjc is a dimer with a large DNA-binding surface consisting of numerous basic residues surrounding the metal-binding residues of the active sites. Residues critical for catalysis, identified on the basis of sequence comparisons and site-directed mutagenesis studies, are clustered to produce two active sites in the dimer, about 29 A apart, consistent with the requirement for the introduction of paired nicks in opposing strands of the four-way DNA junction substrate. Hjc displays similarity to the restriction endonucleases in the way its specific DNA-cutting pattern is determined but uses a different arrangement of nuclease subunits. Further structural similarity to a broad group of metal/phosphate-binding proteins, including conservation of active-site location, is observed. A high degree of conservation of surface electrostatic character is observed between Hjc and T4-phage endonuclease VII despite a complete lack of structural homology. A model of the Hjc-Holliday junction complex is proposed, based on the available functional and structural data.
Subject(s)
Endodeoxyribonucleases/chemistry , Sulfolobus/enzymology , Amino Acid Sequence , Catalytic Domain , Dimerization , Holliday Junction Resolvases , Models, Molecular , Molecular Sequence DataABSTRACT
The structure of a tetragonal crystal form of thiostrepton has been solved using the anomalous dispersive effects of five S atoms from high-redundancy data collected to 1.33 A resolution at the Cu Kalpha wavelength. Data measured to 1.02 A resolution with a synchrotron source were used for refinement. Details of the molecular structure, intramolecular and intermolecular interactions are given.
Subject(s)
Anti-Bacterial Agents/chemistry , Sulfur/chemistry , Thiostrepton/chemistry , Crystallization , Models, Molecular , Protein ConformationABSTRACT
Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloroflexus aurantiacus, crystallizes in space group P6(4)22 (a=b=115.7 A, c=54.6 A). The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was refined at 1.55 A resolution. The molecular model comprises 139 amino acid residues, one Cu, 247 H(2)O molecules, one Cl(-) and two SO(4)(2-). The final residual and estimated standard uncertainties are R=0.198, ESU=0.076 A for atomic coordinates and ESU=0.05 A for Cu---ligand bond lengths, respectively. The auracyanin B molecule has a standard cupredoxin fold. With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.
Subject(s)
Bacterial Proteins/chemistry , Chlorobi/chemistry , Metalloproteins/chemistry , Amino Acid Sequence , Azurin/chemistry , Bacterial Proteins/metabolism , Binding Sites , Copper/metabolism , Crystallization , Crystallography, X-Ray , Evolution, Molecular , Histidine/metabolism , Metalloproteins/metabolism , Models, Molecular , Molecular Sequence Data , Phylogeny , Plastocyanin/chemistry , Protein Folding , Protein Structure, Secondary , Sequence AlignmentABSTRACT
The active conformation of the dimeric cofactor-dependent phosphoglycerate mutase (dPGM) from Escherichia coli has been elucidated by crystallographic methods to a resolution of 1.25 A (R-factor 0.121; R-free 0.168). The active site residue His(10), central in the catalytic mechanism of dPGM, is present as a phosphohistidine with occupancy of 0.28. The structural changes on histidine phosphorylation highlight various features that are significant in the catalytic mechanism. The C-terminal 10-residue tail, which is not observed in previous dPGM structures, is well ordered and interacts with residues implicated in substrate binding; the displacement of a loop adjacent to the active histidine brings previously overlooked residues into positions where they may directly influence catalysis. E. coli dPGM, like the mammalian dPGMs, is a dimer, whereas previous structural work has concentrated on monomeric and tetrameric yeast forms. We can now analyze the sequence differences that cause this variation of quaternary structure.
Subject(s)
Escherichia coli/enzymology , Phosphoglycerate Mutase/chemistry , Amino Acid Sequence , Binding Sites , Histidine/metabolism , Models, Molecular , Molecular Sequence Data , Phosphoglycerate Mutase/metabolism , Phosphorylation , Protein Folding , Protein Structure, Quaternary , Sequence Homology, Amino AcidABSTRACT
Tryparedoxin peroxidase (TryP) is a recently discovered 2Cys-peroxiredoxin involved in defence against oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata TryP, in the reduced state, has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises a decamer with 52 symmetry, ten chloride ions with 23 water molecules and has been refined, using data to 3.2 A resolution (1 A=0.1 nm), to an R-factor and R(free) of 27.3 and 28.6 %, respectively. Secondary structure topology places TryP along with tryparedoxin and glutathione peroxidase in a distinct subgroup of the thioredoxin super-family. The molecular details at the active site support ideas about the enzyme mechanism and comparisons with an oxidised 2Cys-peroxiredoxin reveal structural alterations induced by the change in oxidation state. These include a difference in quaternary structure from dimer (oxidised form) to decamer (reduced form). The 2Cys-peroxiredoxin assembly may prevent indiscriminate oligomerisation, localise ten peroxidase active sites and contribute to both the specificity of reduction by the redox partner tryparedoxin and attraction of peroxides into the active site.
Subject(s)
Crithidia fasciculata/enzymology , Peroxidases/chemistry , Peroxidases/metabolism , Protozoan Proteins , Amino Acid Sequence , Animals , Binding Sites , Catalysis , Crystallography, X-Ray , Dimerization , Disulfides/metabolism , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Structure, Quaternary , Protein Structure, Secondary , Rats , Sequence Alignment , Structure-Activity RelationshipABSTRACT
The Pr(i) sidechains of two adjacent valine residues, V8 and V44, define the surface of the rubredoxin from Clostridium pasteurianum and control access to its Fe(S-Cys)4 active site. To assess the effect of systematic change of the steric bulk of the alkyl sidechains, eight single and three double mutant proteins have been isolated which vary G (H), A (Me), V (Pr(i)), L (Bu(i)) and I (Bu(s)) at those positions. X-ray crystal structures of the Fe(III) forms of the V44A and V44I proteins are reported. Positive shifts in reversible potential of up to 116 mV are observed and attributed to increased polarity around the Fe(S-Cys)4 site induced by (1) changes in protein backbone conformation driven by variation of the steric demands of the sidechain substituents and (2) changes in solvent access to the side-chains of ligands C9 and C42. Data for the V44A mutant show that a minor change in the steric requirements of a surface residue can introduce a NH...Sgamma hydrogen bond at the active site and lead to a shift in potential of + 50 mV.
Subject(s)
Clostridium/chemistry , Rubredoxins/genetics , Valine/genetics , Base Sequence , Crystallography, X-Ray , DNA Primers , Mutagenesis , Protein Conformation , Rubredoxins/chemistry , SolventsSubject(s)
Biotechnology , Genetic Engineering , Policy Making , Politics , Commerce , Consumer Advocacy , Crops, Agricultural/genetics , Global Health , Mass Media , Public PolicyABSTRACT
BACKGROUND: Trypanothione reductase (TR) helps to maintain an intracellular reducing environment in trypanosomatids, a group of protozoan parasites that afflict humans and livestock in tropical areas. This protective function is achieved via reduction of polyamine-glutathione conjugates, in particular trypanothione. TR has been validated as a chemotherapeutic target by molecular genetics methods. To assist the development of new therapeutics, we have characterised the structure of TR from the pathogen Trypanosoma cruzi complexed with the substrate trypanothione and have used the structure to guide database searches and molecular modelling studies. RESULTS: The TR-trypanothione-disulfide structure has been determined to 2.4 A resolution. The chemical interactions involved in enzyme recognition and binding of substrate can be inferred from this structure. Comparisons with the related mammalian enzyme, glutathione reductase, explain why each enzyme is so specific for its own substrate. A CH***O hydrogen bond can occur between the active-site histidine and a carbonyl of the substrate. This interaction contributes to enzyme specificity and mechanism by producing an electronic induced fit when substrate binds. Database searches and molecular modelling using the substrate as a template and the active site as receptor have identified a class of cyclic-polyamine natural products that are novel TR inhibitors. CONCLUSIONS: The structure of the TR-trypanothione enzyme-substrate complex provides details of a potentially valuable drug target. This information has helped to identify a new class of enzyme inhibitors as novel lead compounds worthy of further development in the search for improved medicines to treat a range of parasitic infections.
Subject(s)
Glutathione/analogs & derivatives , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/metabolism , Spermidine/analogs & derivatives , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Dimerization , Drug Design , Flavin-Adenine Dinucleotide/metabolism , Glutathione/chemistry , Glutathione/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , NADH, NADPH Oxidoreductases/antagonists & inhibitors , Protein Conformation , Spermidine/chemistry , Spermidine/metabolism , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacologyABSTRACT
The mucopolysaccharidoses (MPS) are a group of multiple pathology disorders which are part of a larger group of genetic diseases known as lysosomal storage disorders. Enzyme replacement therapy (ERT) has been developed as a therapy for MPS patients. However, immune responses to ERT have been reported in MPS animal models and in human Gaucher patients. Antibodies can have adverse effects during ERT, which include hypersensitivity/anaphylactic reactions, enzyme inactivation, and enzyme degradation. This study aimed to characterize the immune response to ERT in a feline model of MPS VI, by defining the epitope reactivity of cat plasma antibody against human recombinant N-acetylgalactosamine 4-sulfatase (4-sulfatase) replacement protein. For MPS VI cat plasma, antibody reactivity was observed prior to ERT, with distinct regions of 4-sulfatase linear sequence displaying low affinity antibody reactivity. There was an increase in antibody titer to 4-sulfatase for MPS VI cats post-ERT, with the majority of the immune response detected to linear sequence epitopes. One cat displayed a high titer and high affinity epitope reactivity following prolonged exposure (>/=9 months) to the replacement protein. MPS VI cats on shorter term ERT (3 months) showed high titers to 4-sulfatase and similar patterns of epitope reactivity, but lower affinity antibody reactivity, when compared to the latter cat. This study reports the linear amino acid sequence reactivity and nature of the immune response produced to 4-sulfatase before and after ERT. The monitoring of antibody production during replacement therapy is an important consideration for patient management, as high titer antibodies can affect the efficacy of therapy.
Subject(s)
Antibodies/blood , Epitopes/immunology , Mucopolysaccharidosis VI/drug therapy , Mucopolysaccharidosis VI/immunology , N-Acetylgalactosamine-4-Sulfatase/immunology , N-Acetylgalactosamine-4-Sulfatase/therapeutic use , Animals , Cats , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Humans , Models, Molecular , Protein Denaturation , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Treatment OutcomeABSTRACT
The crystal structure of the 'blue' copper protein plastocyanin from the cyanobacterium Phormidium laminosum has been solved and refined using 2.8 A X--ray data. P. laminosum plastocyanin crystallizes in space group P43212 with unit-cell dimensions a = 86.57, c = 91.47 A and with three protein molecules per asymmetric unit. The final residual R is 19.9%. The structure was solved using molecular replacement with a search model based on the crystal structure of a close homologue, Anabaena variabilis plastocyanin (66% sequence identity). The molecule of P. laminosum plastocyanin has 105 amino-acid residues. The single Cu atom is coordinated by the same residues - two histidines, a cysteine and a methionine - as in other plastocyanins. In the crystal structure, the three molecules of the asymmetric unit are related by a non-crystallographic threefold axis. A Zn atom lies between each pair of neighbouring molecules in this ensemble, being coordinated by a surface histidine residue of one molecule and by two aspartates of the other.
Subject(s)
Cyanobacteria/chemistry , Plastocyanin/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Zinc/chemistryABSTRACT
The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.
Subject(s)
Aminopeptidases/metabolism , Escherichia coli/enzymology , Protein Conformation , Aminopeptidases/chemistry , Aminopeptidases/genetics , Binding Sites , Hydrogen-Ion Concentration , Molecular Sequence Data , Structure-Activity Relationship , Substrate SpecificityABSTRACT
BACKGROUND: . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS: . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS: . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.
Subject(s)
Chondro-4-Sulfatase/chemistry , Lysosomes/enzymology , Protein Conformation , Alkaline Phosphatase/chemistry , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Chondro-4-Sulfatase/antagonists & inhibitors , Chondro-4-Sulfatase/deficiency , Chondro-4-Sulfatase/genetics , Consensus Sequence , Cricetinae , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Glycosylation , Humans , Models, Molecular , Molecular Sequence Data , Mucopolysaccharidosis VI/enzymology , Mucopolysaccharidosis VI/genetics , Multigene Family , Point Mutation , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Vanadates/metabolism , Vanadates/pharmacologyABSTRACT
A comprehensive patient information datafile of 320 topics has been developed, subserving the domains of medicine, surgery, gynaecology and paediatrics. The system was designed as loose-leaf sheets capable of being photocopied, as well as a computer-based datafile. In a four-practice study, 73% of consecutive general practice attenders could be issued with the relevant disorder or procedure information sheet. With a questionnaire return rate of 79%, 886 patients rated the three criteria of readability, understandability and usefulness of their leaflets as very or quite easy and very or quite useful in more than 94% of instances. This system could be a valuable adjunct to patient education in both general and hospital practice settings.
Subject(s)
Family Practice , Information Services , Health Education , Humans , Patient Education as Topic , Patient Satisfaction , United KingdomABSTRACT
Trypanothione reductase (TR) is an NADPH-dependent flavoprotein unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved trypanocidal drugs. We present details of the structure of TR from the human pathogen Trypanosoma cruzi, the agent responsible for Chagas' disease or South American trypanosomiasis. The structure has been solved by molecular replacement, using as the starting model the structure of the enzyme from the nonpathogenic Crithidia fasciculata, and refined to an R-factor of 18.9% for 53,868 reflections with F > or = sigma F between 8.0 and 2.3 A resolution. The model comprises two subunits (968 residues), two FAD prosthetic groups, two maleate ions, and 419 water molecules. The accuracy and geometry of the enzyme model is improved with respect to the C. fasciculata enzyme model. The new structure is described and specific features of the enzyme involved in substrate interactions are compared with previous models of TR and related glutathione reductases from human and Escherichia coli. Structural differences at the edge of the active sites suggest an explanation for the differing specificities toward glutathionylspermidine disulfide.