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D W Rice

Publications and source records attributed to D W Rice.

At least 19 recordsLinked to original sources
PubMed2006

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Amino Acid Sequence↗
PubMed2003

Delivery of antimicrobials into parasites.

To eliminate apicomplexan parasites, inhibitory compounds must cross host cell, parasitophorous vacuole, and parasite membranes and cyst walls, making delivery challenging. Here, we show that short oligomers of arginine enter Toxoplasma gondii tachyzoites and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP reductase (ENR), conjugated to arginine oligomers enters extracellular tachyzoites, host cells, tachyzoites inside parasitophorous vacuoles within host cells, extracellular bradyzoites, and bradyzoites within cysts. We identify, clone, and sequence T. gondii enr and produce and characterize enzymatically active, recombinant ENR. This enzyme has the requisite amino acids to bind triclosan. Triclosan released after conjugation to octaarginine via a readily hydrolyzable ester linkage inhibits ENR activity, tachyzoites in vitro, and tachyzoites in mice. Delivery of an inhibitor to a microorganism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to sequestered parasites, a model system to characterize transport across multiple membrane barriers and structures, a widely applicable paradigm for treatment of active and encysted apicomplexan and other infections, and a generic proof of principle for a mechanism of medicine delivery.

Amino Acid Sequence↗
PubMed2003

Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa.

Current treatments for diseases caused by apicomplexan and trypanosomatid parasites are inadequate due to toxicity, the development of drug resistance and an inability to eliminate all life cycle stages of these parasites from the host. New therapeutics agents are urgently required. It has recently been demonstrated that type II fatty acid biosynthesis occurs in the plastid of Plasmodium falciparum and Toxoplasma gondii and inhibitors of this pathway such as triclosan and thiolactomycin restrict their growth. Furthermore, Trypanosoma brucei has recently been demonstrated to use type II fatty acid biosynthesis for myristate synthesis and to be susceptible to thiolactomycin. As this pathway is absent from mammals, it may provide an excellent target for novel antimicrobial agents to combat these diverse parasites. Leishmania and Trypanosoma parasites produce ergosterol-related sterols by a biosynthetic pathway similar to that operating in pathogenic fungi and their growth is susceptible to sterol biosynthesis inhibitors. Thus, inhibition of squalene 2,3-epoxidase by terbinafine, 14alpha-methylsterol 14-demethylase by azole and triazole compounds and delta(24)-sterol methyl transferase by azasterols all cause a depletion of normal sterols and an accumulation of abnormal amounts of sterol precursors with cytostatic or cytoxic consequences. However, Leishmania parasites can survive with greatly altered sterol profiles induced by continuous treatment with low concentrations of some inhibitors and they also have some ability to utilise and metabolise host sterol. These properties may permit the parasites to evade treatment with sterol biosynthesis inhibitors in some clinical situations and need to be taken into account in the design of future drugs.

Animals↗
PubMed2003

Total and water-soluble phosphorus in broiler litter over three flocks with alum litter treatment and dietary inclusion of high available phosphorus corn and phytase supplementation.

Three pen trials were conducted to determine the main effect of alum addition to litter on form of poultry litter P using a 2 x 2 factorial structure of the subunit treatments: diets including high available phosphorus/low phytate corn (HAPC) and phytase (PHYT). Male broilers (1,760 per flock) were grown to 42 d having starter diets with 0.45% available P and grower diets with 0.35% available P. In the first trial, total litter P (tP) was greatest for the yellow dent corn (YDC) diet (12 g/kg) and least for the HAPC and PHYT combination (H&P) diet (6.9 g/kg) with the individual PHYT and HAPC diets falling in between at 9.1 g/kg and 9.4 g/kg tP. Also in the first trial, the litter water-soluble P (wP) was highest for PHYT (2.8 g/kg), least for the HAPC and H&P diets (1.5 g/kg) with the YDC diet falling between (2.2 g/kg). Alum was added to the litter after the first experiment. In the second and third experiments, alum inclusion significantly reduced the wP when compared with the treatments with no alum. In the third trial, the least wP was present in the alum-HAPC treatment. Phytase, YDC, and HAPC diets with no alum litter treatment generated the most wP. Since these diets appear to have little or no difference with respect to quantity of wP, this work suggests that form of litter P generated by alternative diets should be considered as criteria when attempting to reduce P in broiler litter applied to land.

6-Phytase↗
PubMed2002

Crystallization and preliminary X-ray analysis of the ytxM gene product from Bacillus subtilis.

The ytxM gene product from Bacillus subtilis has been cloned, expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Multiple-wavelength anomalous dispersive X-ray data have been collected to 2.0 A resolution on a single selenomethionine-incorporated crystal. This crystal belongs to the primitive orthorhombic system, with approximate unit-cell parameters a = 44.3, b = 90.9, c = 136.1 A, alpha = beta = gamma = 90 degrees and two monomers in the asymmetric unit.

Amino Acid Sequence↗
PubMed2002

Insights into enzyme evolution revealed by the structure of methylaspartate ammonia lyase.

Methylaspartate ammonia lyase (MAL) catalyzes the magnesium-dependent reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid. The 1.3 A MAD crystal structure of the dimeric Citrobacter amalonaticus MAL shows that each subunit comprises two domains, one of which adopts the classical TIM barrel fold, with the active site at the C-terminal end of the barrel. Despite very low sequence similarity, the structure of MAL is closely related to those of representative members of the enolase superfamily, indicating that the mechanism of MAL involves the initial abstraction of a proton alpha to the 3-carboxyl of (2S,3S)-3-methylasparic acid to yield an enolic intermediate. This analysis resolves the conflict that had linked MAL to the histidine and phenylalanine ammonia lyase family of enzymes.

Amino Acid Sequence↗
PubMed2001

Crystallization and preliminary X-ray analysis of glucose dehydrogenase from Haloferax mediterranei.

Glucose dehydrogenase (E.C. 1.1.1.47; GlcDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized by the addition of 8 M urea and refolded by rapid dilution. The protein has been purified by conventional techniques and crystallized by the hanging-drop vapour-diffusion method using sodium citrate as the precipitant. Two crystal forms representing the free enzyme and the binary complex with NADP(+) grow under these conditions. Crystals of form I diffract to beyond 3.5 A resolution and belong to the hexagonal space group P622, with unit-cell parameters a = b = 89.1, c = 214.6 A, alpha = beta = 90, gamma = 120 degrees. Crystals of form II diffract to greater than 2.0 A and belong to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 61.8, b = 110.9, c = 151.7 A, alpha = beta = gamma = 90 degrees. Calculated values for V(M) and consideration of the packing for both crystal forms suggests that the asymmetric units in both crystal forms contain a monomer.

Crystallization↗
PubMed2001

Crystallization and preliminary X-ray analysis of Citrobacter amalonaticus methylaspartate ammonia lyase.

Methylaspartate ammonia lyase (MAL) catalyses the reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid. Crystals of Citrobacter amalonaticus MAL have been obtained by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. Three crystal forms were obtained from identical crystallization conditions, two of which (forms A and B) diffract to high resolution, whilst the third form diffracted poorly. Crystals of form A diffract to beyond 2.1 A and have been characterized as belonging to one of the enantiomorphic space groups P4(1)22 or P4(3)22, with unit-cell parameters a = b = 66.0, c = 233.1 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit. Crystals of form B diffract to beyond 1.5 A and belong to space group C222, with unit-cell parameters a = 128.3, b = 237.4, c = 65.8 A, alpha = beta = gamma = 90 degrees and a dimer in the asymmetric unit. Determination of the structure of MAL will be an important step in resolving current conflicts concerning the enzyme mechanism which differ between one which places MAL as a member of the superfamily of ammonia lyases whose catalytic activity requires a cofactor formed by post-translational modification of the enzyme and another which links MAL to the enolase superfamily.

Ammonia-Lyases↗
PubMed2001

The 1.2 A structure of a novel quorum-sensing protein, Bacillus subtilis LuxS.

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione. The crystal structure of Bacillus subtilis LuxS has been determined at 1.2 A resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 and 2.3 A resolution, respectively. These structures show that LuxS is a homodimer with an apparently novel fold based on an eight-stranded beta-barrel, flanked by six alpha-helices. Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits. S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme-bound zinc ion. Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125-129 and those at the N terminus. The structure contains an oxidised cysteine residue in the active site whose role in the biological process of LuxS has not been determined. The autoinducer-2 signalling pathway has been linked to aspects of bacterial virulence and pathogenicity. The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics.

Amino Acid Sequence↗
PubMed2001

The crystal structure of Thermotoga maritima maltosyltransferase and its implications for the molecular basis of the novel transfer specificity.

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from alpha-1,4-linked glucans or malto-oligosaccharides to other alpha-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (beta/alpha)(8) barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 A and 2.1 A resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (beta/alpha)(8) barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites -2 and -1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the -2 subsite by the side-chain of Lys151 and the stretch of residues 314-317, providing an explanation for the strict transfer specificity of MTase.

Binding Sites↗
PubMed2001

The structure and domain organization of Escherichia coli isocitrate lyase.

Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 A resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle.

Alanine↗
PubMed2001

Cloning, purification, crystallization and preliminary crystallographic analysis of Bacillus subtilis LuxS.

LuxS of Bacillus subtilis is a member of a novel family of proteins with a potential role in quorum sensing, controlling important aspects of cellular physiology in a range of microbial species. B. subtilis luxS was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to one of the enantiomorphic space groups P6(1)22 or P6(5)22, with approximate unit-cell parameters a = b = 63.6, c = 151.5 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.5 A(3) Da(-1). The crystals diffract X-rays to at least 1.55 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinants of function of this class of proteins, for which no structures are currently available.

Bacillus subtilis↗
PubMed2001

The preparation and crystallization of Fab fragments of a family of mouse esterolytic catalytic antibodies and their complexes with a transition-state analogue.

The Fab fragments of a family of mouse esterolytic monoclonal antibodies MS6-12, MS6-126 and MS6-164 have been obtained by digestion of whole antibodies with papain, purified and crystallized in a range of different forms either alone or in complex with a transition-state analogue. The crystals diffract X-rays to resolutions between 2.1 and 1.2 A and are suitable for structural studies. The determination of these structures could be important in understanding the different catalytic power of each of these related catalytic antibodies.

Animals↗
PubMed2001

Crystallization and preliminary X-ray crystallographic studies on 4-alpha-glucanotransferase from Thermotoga maritima.

Thermotoga maritima 4-alpha-glucanotransferase (GTase), a 52 kDa molecular-weight amylolytic enzyme, has been crystallized by the hanging-drop vapour-diffusion method using PEG monomethylether 5000 as a precipitating agent. A complete data set has been collected to 2.6 A resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 92.6, b = 180.3, c = 199.2 A.

Crystallography, X-Ray↗
PubMed2001

A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase.

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.

Binding Sites↗
PubMed2001

Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus.

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group I4(1)22, with unit-cell parameters a = b = 104.5, c = 245.1 A. Consideration of the values of V(M) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a dimer. Examination of the behaviour of the protein on gel-filtration columns and analysis of the self-rotation function suggests that the molecule is an octamer in solution at around pH 5. Determination of the structure of this protein will provide insights into the mechanisms responsible for DNA-protein recognition at high temperature and into how the regulatory properties of the Lrp family are modified by the presence or absence of effector molecules.

Archaeal Proteins↗
PubMed2001

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.

Amino Acid Sequence↗