Biomedical subjects

W N Hunter

Publications and source records attributed to W N Hunter.

At least 37 records · Page 2Linked to original sources

PubMed1999

Crystallization of recombinant Leishmania major pteridine reductase 1 (PTR1).

The enzyme pteridine reductase (PTR1) has recently been discovered in the protozoan parasite Leishmania and validated as a target for therapeutic intervention. PTR1 is responsible for the salvage of pteridines and also contributes to antifolate drug resistance. Structural analysis, in combination with ongoing biochemical characterization will assist the elucidation of the structure-activity relationships of this important enzyme and support a structure-based approach to discover novel inhibitors. Recombinant L. major PTR1 has been purified from an Escherichia coli expression system and used in crystallization experiments. Orthorhombic crystals have been obtained and data to 2.8 A has been measured. The space group is P2(1)2(1)2 or P2(1)2(1)2(1) with unit-cell dimensions of a = 103.9, b = 134.7, c = 96.2 A. One homotetramer, of molecular mass approximately 120 kDa, probably constitutes the asymmetric unit and gives a Matthews coefficient, V(m), of 2.8 A(3) Da(-1) and 56% solvent volume. Self-rotation function calculations show a single well defined non-crystallographic twofold axis with features that might represent additional elements of non-crystallographic symmetry. The detail of exactly what constitutes the asymmetric unit will be resolved by structure determination.

Animals↗

PubMed1998

Crystallization and preliminary X-ray diffraction studies of 6-phosphogluconate dehydrogenase from Lactococcus lactis.

6-Phosphogluconate dehydrogenase is one of the seven enzymes involved in the pentose phosphate pathway. Crystals of a mammalian and a protozoan enzyme have been obtained previously and structures determined. It is reported here that a bacterial 6-phosphogluconate dehydrogenase, from Lactococcus lactis, has been purified and used in crystallization trials. Large prisms suitable for a detailed structural analysis have been obtained and characterized as orthorhombic, space group F222, with a = 70.4, b = 105.7, c = 474.6 A. Diffraction has been observed to 2.2 A resolution using synchrotron radiation. Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationships of this enzyme.

Bacterial Proteins↗

PubMed1997

Structure of ferric soybean leghemoglobin a nicotinate at 2.3 A resolution.

Soybean leghemoglobin a is a small (16 kDa) protein facilitating the transport of O(2) to respiring N(2)-fixing bacteria at low free-O(2) tension. The crystal structure of soybean ferric leghemoglobin a nicotinate has been refined at 2.3 A resolution. The final R factor is 15.8% for 6877 reflections between 6.0 and 2.3 A. The structure of soybean leghemoglobin a (143 residues) is closely similar to that of lupin leghemoglobin II (153 residues), the proteins having 82 identical residues when the sequences are aligned. The new structure provides support for the conclusion that the unique properties of leghemoglobin arise principally from a heme pocket considerably larger and more flexible than that of myoglobin, a strongly ruffled heme group, and a proximal histidine orientation more favourable to ligand binding.

Journal Article↗

PubMed1997

A structure-based approach to drug discovery; crystallography and implications for the development of antiparasite drugs.

Advances in the life and physical sciences have enabled us to characterize the 3-dimensional structure and the biochemical or biological activity of both small and large molecules. The use of structural chemistry to assist understanding of biological activity provides information relevant to the design, development or identification of new pharmaceuticals. This structure based approach has become an important component of drug research and is in widespread use by the major pharmaceutical companies. A brief historical introduction, to convey how this area of science has reached the present stage, is given. The basis of the structural approach to understanding the chemistry of small and large molecule biological activity is outlined with an emphasis on the use of results derived from X-ray diffraction methods. Developments in other areas are discussed to emphasize the multidisciplinary nature of this research and the benefits of combining different methods. Examples of protein crystallographic studies in the area of molecular parasitology, some of which are directly relevant to antiparasite drug design, are presented. The characterization of the enzyme trypanothione reductase, a project which has benefited from many of the recent developments, is detailed. Future challenges and difficulties, both scientific and economic, are discussed.

Antiparasitic Agents↗

PubMed1996

The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold.

BACKGROUND: [corrected] Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution. The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Amino Acid Sequence↗

PubMed1996

The crystal structure analysis of d(CGCGAASSCGCG)2, a synthetic DNA dodecamer duplex containing four 4'-thio-2'-deoxythymidine nucleotides.

The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG), where S = 4'-thio-2'-deoxythymidine, has converged at R=0.201 for 2605 reflections with F > 2sigma(F) in the resolution range 8.0-2.4 A for a model consisting of the dodecamer duplex and 66 water molecules. A comparison of its structure with that of the native dodecamer d(CGCGAATTCGCG) has revealed that the major differences between the two structures is a change in the conformation of the sugar-phosphate backbone in the regions at and adjacent to the positions of the modified nucleosides. Examination of the fine structural parameters for each of the structures reveals that the thiosugars adopt a C3'-exo conformation in d(CGCGAASSCGCG), rather than the approximate C1'-exo conformation found for the analogous sugars in the structure of d(CGCGAATTCGCG). The observed differences in structure between the two duplexes may help to explain the enhanced resistance to nuclease digestion of synthetic oligonucleotides containing 4'-thio-2'-deoxynucleotides.

Base Sequence↗

PubMed1996

MAD Phasing Strategies Explored with a Brominated Oligonucleotide Crystal at 1.65A Resolution.

The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.

Journal Article↗

PubMed1996

The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 A resolution.

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.

Amino Acid Sequence↗

PubMed1995

p-(4,6-Diamino-s-triazin-2-yl)aminophenylarsonic acid dihydrate.

The structure of the title compound C9 H11 AsN6O3-2H2O, contains a planar organic moiety bonded to pentavalent arsenic. The lattice is stabilized by a combination of aromatic stacking interactions and by a hydrogen-bonding network involving water molecules of crystallization.

Arsenicals↗

PubMed1995

Additional crystal forms of the E. coli class II fructose-1,6-bisphosphate aldolase.

We have obtained two additional crystal forms of the metal-dependent class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of elongated plates have unit-cell dimensions a = 73.4, b = 120.0, c = 190.1 A, orthorhombic space group P2(1)2(1)2(1). Monoclinic prisms have unit-cell dimensions a = 67.7, b = 104.3, c = 52.8 A, beta = 105 degrees, space group P2(1). Diffraction to slightly better than 3.0 A, has been observed for both forms using in-house and synchrotron facilities. These crystal forms may aid the structure solution of this enzyme by presenting additional forms for heavy-atom derivatization. These forms have multiple copies of the enzyme in the asymmetric unit and averaging methods might also be useful in the analysis.

Journal Article↗

PubMed1995

Evidence for two distinct azurins in Alcaligenes xylosoxidans (NCIMB 11015): potential electron donors to nitrite reductase.

We have isolated two type 1 copper-containing proteins (M(r) approximately 13K) from Alcaligenes xylosoxidans (NCIMB 11015) grown under denitrifying conditions. Amino acid sequence analysis of these two proteins shows one to be the previously identified azurin (Ambler, 1971), which we shall call azurin I, and the other to be a related, but previously undescribed, blue copper protein which we show to also be an azurin and propose to call azurin II. Thus, NCIMB 11015 becomes the second system where two distinct azurins are found, the other being Methylomonas J (Ambler & Tobari, 1989). On isoelectric focusing, azurin I migrates very similarly to the previously identified azurin from this organism while azurin II migrates similarly to azurin purified from Alcaligenes denitrificans NCTC 8582. The sequence of azurin II is 33% different than the azurin I sequence but is only 11% different than the azurin from Alcaligenes denitrificans NCTC 8582. Optical spectra for the two proteins are very similar with epsilon mM values of 6.27 and 5.73 mM-1 cm-1 for azurin I and II, respectively, at lambda max approximately 620 nm. The 291 nm shoulder normally ascribed to the hydrophobic nature of tryptophan 48 is clearly observed in azurin I but is missing in azurin II. Amino acid analysis confirms that this tryptophan is missing in azurin II. Azurin I and azurin II show essentially the same redox potential of 305 +/- 10 mV at pH 7.5 and are equally effective electron donors to the purified dissimilatory nitrite reductase of Alc. xylosoxidans in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗

PubMed1995

A comparison of two independently determined structures of trypanothione reductase from Crithidia fasciculata.

The enzyme trypanothione reductase (TR) is unique to trypanosomes and leishmania parasites, the causal agents of several important medical and veterinary tropical diseases. TR helps regulate the intracellular reducing environment of the parasite and it has been identified as a target for developing novel chemotherapeutic agents by structure-aided drug design. For this purpose it is essential to have confidence in the structural detail of the molecular target. Two independent studies of Crithidia fasciculata TR at medium resolution, in different space groups have afforded an opportunity to assess the reliability of the models. We summarize the important methodological details of each analysis and present a comparison of the geometry, thermal parameters and three-dimensional structure of the models. Particular attention has been paid to the disulfide substrate-binding site which is the area of most interest with respect to enzyme inhibition. The comparison has shown that the structures agree closely with Calpha atoms superposing with an r.m.s. of less than 0.5 A. The consistency of the models gives a high level of confidence that they are suitable for computer-aided drug design. The conformation of many side chains in the active site, in particular the catalytic residues, are well conserved in both structures. However, the comparison indicates a difference in the conformation of Trp21 and Met113 which together form a hydrophobic patch on the rim of the active-site cleft and interact with the spermidine moiety of the substrate. Consideration of the electron-density maps together with the structural comparison indicates that there is some conformational flexibility in this region of the active site. This heterogeneity may be used in the recognition of the substrate by the enzyme and should be considered when mapping out the size, shape and chemical properties of the active site.

Journal Article↗

PubMed1995

A trigonal form of the idarubicin:d(CGATCG) complex; crystal and molecular structure at 2.0 A resolution.

The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.

Base Sequence↗

PubMed1995

Self-association of a DNA loop creates a quadruplex: crystal structure of d(GCATGCT) at 1.8 A resolution.

BACKGROUND: The flexibility of DNA enables it to adopt three interconvertible types of duplex termed the A-, B- and Z-forms. It can also produce hairpin loops, triplex structures and guanine-rich quadruplex structures. Conformational flexibility assists in the tight packaging of DNA, for example in chromosomes. This is important given the large quantity of genetic information that must be packaged efficiently. Moreover, the ability of DNA to specifically self-associate or interact with complementary sequences is fundamental to many biological processes. Structural studies provide information about DNA conformation and DNA-DNA interactions and suggest features that might be relevant to how the molecule performs its biological role. RESULTS: We have characterized the structure of a synthetic heptanucleotide that folds into a novel loop structure. The loop is stabilized by association with a cation, by intra-strand hydrogen bonds between guanine and cytosine that are distinct from the normal Watson-Crick hydrogen bonds, and by van der Waals interactions. Two loops associate through the formation of four G.C pairs that exhibit pronounced base-stacking interactions. The formation of a symmetric A.A base pair further stabilizes loop dimerization. Stacking of the A.A pair on a symmetry-related A.A pairing assists the formation of a four-stranded assembly. A T.T pairing is also observed between symmetry-related loops. CONCLUSIONS: This analysis provides a rare example of an experimentally determined non-duplex DNA structure. It provides conformational detail relevant to the tight packaging or folding of a DNA strand and illustrates how a cation might modulate phosphate-phosphate repulsion in a tightly packed structure. The observation of base quartets involving G.C base pairs suggests a further structure to be considered in DNA-DNA interactions. The structure also provides detailed geometries for A.A and T.T base pairs.

Base Composition↗

PubMed1995

Do C-H...O hydrogen bonds contribute to the stability of nucleic acid base pairs?

The possible formation of inter-base C-H.O hydrogen bonds in A.T, A.U and certain non-Watson-Crick base pairs is examined. A geometrical analysis in conjunction with implications for the thermodynamic stability of the base pairs suggests that C-H.O hydrogen bonds could form in nucleic acid base pairs. They may alleviate destabilizing interactions that would arise if an unsatisfied hydrogen-bond acceptor were present and mediate secondary hydrogen-bonding effects in these base pairs.

Journal Article↗

PubMed1995

Rational drug design: a multidisciplinary approach.

The pharmaceutical industry developed spasmodically and, in its early years, was shaped by several notable events and scientists. Paul Ehrlich, whose studies initiated the field of chemotherapy, was one of the major contributors. From early experiments on the efficacy of the biological stain methylene blue as an anti-malarial, Ehrlich realized that small molecules could be used to treat infections. Further studies identified trypan dyes and arsenical compounds as treatments of trypanosomal infection, and the arsenical compounds were found to be effective against syphilis. Ehrlich's experience of the chemical industry, in particular the chemistry of dyes, led to the realization that synthetic molecules could be medically useful and provided a powerful stimulus to pharmaceutical research.

Drug Design↗

PubMed1994

Refined structure of concanavalin A complexed with methyl alpha-D-mannopyranoside at 2.0 A resolution and comparison with the saccharide-free structure.

The three-dimensional structure of the complex between methyl alpha-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P2(1)2(1)2(1), a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0sigma(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A(2) when it binds to the protein. This work builds upon the earlier studies of Derewenda et al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan & Campbell (1989). EMBO J. 8, 2198-2193] at 2.9 A resolution, which was the first detailed study of lectin-saccharide interactions.

Journal Article↗

PubMed1994

Crystal structure of a DNA duplex containing 8-hydroxydeoxyguanine-adenine base pairs.

The crystal structure of the oligonucleotide d(CGCAAATTO8GGCG), containing the chemically modified base 8-hydroxydeoxyguanine (O8G), has been determined at 2.5-A resolution and refined to a crystallographic R-factor of 16.8%. The B-type DNA helix contains standard Watson-Crick base pairs except at the mismatch sites, where O8G adopts a syn conformation and forms hydrogen bonds to adenine in the anti conformation. The thermodynamic stability of the duplex was found by UV melting techniques to be intermediate between the native oligonucleotide d(CGCAAATTTGCG) and an oligonucleotide containing A.G mispairs d(CGCAAATTGGCG). Comparison of the structure of the O8G(syn).A(anti) base pair with those of Watson-Crick base pairs has given a reason why O8G.A base pairs are not well repaired by DNA proofreading enzymes.

8-Hydroxy-2'-Deoxyguanosine↗