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1.
Biochim Biophys Acta ; 1794(10): 1510-6, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19595801

ABSTRACT

Bacillus anthracis is a Gram-positive spore-forming bacterium that is the causative agent of anthrax disease. The use of anthrax as a bioweapon has increased pressure for the development of an effective treatment. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthetic pathway yielding two essential bacterial metabolites, meso-diaminopimelate (DAP) and (S)-lysine. DHDPS is therefore a potential antibiotic target, as microbes require either lysine or DAP as a component of the cell wall. This paper is the first biochemical description of DHDPS from B. anthracis. Enzyme kinetic analyses, isothermal titration calorimetry (ITC), mass spectrometry and differential scanning fluorimetry (DSF) were used to characterise B. anthracis DHDPS and compare it with the well characterised Escherichia coli enzyme. B. anthracis DHDPS exhibited different kinetic behaviour compared with E. coli DHDPS, in particular, substrate inhibition by (S)-aspartate semi-aldehyde was observed for the B. anthracis enzyme (K(si(ASA))=5.4+/-0.5 mM), but not for the E. coli enzyme. As predicted from a comparison of the X-ray crystal structures, the B. anthracis enzyme was not inhibited by lysine. The B. anthracis enzyme was thermally stabilised by the first substrate, pyruvate, to a greater extent than its E. coli counterpart, but has a weaker affinity for pyruvate based on enzyme kinetics and ITC studies. This characterisation will provide useful information for the design of inhibitors as new antibiotics targeting B. anthracis.


Subject(s)
Bacillus anthracis/enzymology , Hydro-Lyases/chemistry , Hydro-Lyases/metabolism , Allosteric Regulation , Animals , Bacillus anthracis/drug effects , Bacillus anthracis/genetics , Bacillus anthracis/pathogenicity , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/pharmacology , Enzyme Stability , Escherichia coli/enzymology , Escherichia coli/genetics , Feedback, Physiological , Genes, Bacterial , Humans , Hydro-Lyases/antagonists & inhibitors , Hydro-Lyases/genetics , Hydrophobic and Hydrophilic Interactions , Kinetics , Ligands , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermodynamics
2.
Article in English | MEDLINE | ID: mdl-17329826

ABSTRACT

1,3-Propanediol dehydrogenase (1,3-PD-DH), encoded by the dhaT gene, is a key enzyme in the dissimilation process for converting glycerol to 1,3-propanediol in the human pathogen Klebsiella pneumoniae. Single colourless crystals were obtained from a recombinant preparation of 1,3-propanediol dehydrogenase overexpressed in Escherichia coli. The crystals belong to space group P2(1), with unit-cell parameters a = 91.9, b = 226.6, c = 232.6 A, beta = 92.9 degrees. The crystals probably contain two decamers in the asymmetric unit, with a V(M) value of 3.07 A3 Da(-1) and an estimated solvent content of 59%. Diffraction data were collected to 2.7 A resolution using synchrotron radiation at the ID14-4 beamline of the European Synchrotron Radiation Facility.


Subject(s)
Alcohol Oxidoreductases/chemistry , Bacterial Proteins/chemistry , Crystallography, X-Ray/methods , Klebsiella pneumoniae/enzymology , Alcohol Dehydrogenase , Alcohol Oxidoreductases/isolation & purification , Bacterial Proteins/isolation & purification , Crystallization , Humans , Klebsiella pneumoniae/pathogenicity
3.
Acta Crystallogr D Biol Crystallogr ; 62(Pt 10): 1103-13, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17001088

ABSTRACT

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.


Subject(s)
Cloning, Molecular/methods , Prokaryotic Cells/metabolism , Proteomics/trends , Amino Acid Sequence , Automation , Base Sequence , Escherichia coli/metabolism , Europe , Fermentation , Gene Deletion , Gene Library , Genetic Vectors , Molecular Sequence Data , Protein Folding , Sequence Analysis/instrumentation , Sequence Analysis/methods
4.
Acta Crystallogr D Biol Crystallogr ; 62(Pt 10): 1125-36, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17001090

ABSTRACT

Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.


Subject(s)
Proteins/metabolism , Proteomics/methods , Crystallization , Hydrolysis , Light , Mass Spectrometry , Microscopy, Fluorescence , Models, Molecular , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , Scattering, Radiation , Trypsin , Ultracentrifugation , X-Rays
5.
Acta Crystallogr D Biol Crystallogr ; 62(Pt 10): 1196-207, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17001096

ABSTRACT

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.


Subject(s)
Bacterial Infections/metabolism , Bacterial Proteins/chemistry , Proteomics/methods , Viral Proteins/chemistry , Virus Diseases/metabolism , Animals , Bacterial Infections/microbiology , Humans , Protein Folding , Virus Diseases/virology
6.
Biochem Soc Trans ; 31(Pt 3): 699-702, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12773186

ABSTRACT

Archaeal family-B DNA polymerases possess a novel uracil-sensing mechanism. A specialized pocket scans the template, ahead of the replication fork, for the presence of uracil; on encountering this base, DNA synthesis is stalled. The structural basis for uracil recognition by polymerases is described and compared with other uracil-recognizing enzymes (uridine-triphosphate pyrophophatases and uracil-DNA glycosylases). Remarkably, protein-protein interactions between all three archaeal uracil sensors are observed; possibly the enzymes co-operate to efficiently eliminate uracil from archaeal genomes.


Subject(s)
Archaea/enzymology , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Uracil/metabolism , DNA Repair/genetics , Models, Molecular , Protein Conformation , Pyrophosphatases/chemistry , Pyrophosphatases/metabolism , Substrate Specificity
7.
Nucleic Acids Res ; 28(5): 1059-66, 2000 Mar 01.
Article in English | MEDLINE | ID: mdl-10666444

ABSTRACT

Polymerases from the Pol-I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol-II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. 'Rational' alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild-type Pfu-Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490W), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type. A486, L490 and Y497 are on analpha-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this alpha-helix to a second long helix. None of the four amino acids can contact the dNTP directly. Therefore, the increased selectivity for ddNTPs is likely to arise from two factors: (i) small overall changes in conformation that subtly alter the nucleotide triphosphate binding site such that ddNTPs become favoured; (ii) interference with a conformational change that may be critical both for the polymerisation step and discrimination between different nucleotide triphosphates.


Subject(s)
DNA-Directed DNA Polymerase/metabolism , Nucleotides/metabolism , Pyrococcus furiosus/enzymology , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , Molecular Sequence Data , Mutation , Structure-Activity Relationship
8.
Proc Natl Acad Sci U S A ; 96(16): 9045-50, 1999 Aug 03.
Article in English | MEDLINE | ID: mdl-10430892

ABSTRACT

Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G.C --> A.U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea.


Subject(s)
Cytosine , DNA-Directed DNA Polymerase/metabolism , Mutation , Pyrococcus furiosus/enzymology , Uracil , Base Sequence , DNA Primers , Kinetics , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Surface Plasmon Resonance , Taq Polymerase/metabolism , Templates, Genetic , Thermus/enzymology
9.
J Br Interplanet Soc ; 48(10): 427-34, 1995 Oct.
Article in English | MEDLINE | ID: mdl-11541204

ABSTRACT

The widespread growth of higher plants on Mars following ecopoiesis has often been invoked as a method of generating atmospheric oxygen. However, one issue that has been overlooked in this regard is the fact that terrestrial plants do not thrive under conditions of low oxygen tension. A review of the relevant botanical literature reveals that the high oxygen demands of root respiration could limit the introduction of most plants on Mars until after terraforming has raised the atmospheric pO2 to 20-100 mbar. A variety of physiological strategies are discussed which, if it is possible to implement them in a genetically engineered plant specifically designed for life on Mars, might allow this problem to be overcome.


Subject(s)
Extraterrestrial Environment , Mars , Plant Development , Plant Physiological Phenomena , Anaerobiosis/physiology , Atmosphere/chemistry , Exobiology , Genetic Engineering , Leghemoglobin/metabolism , Oxygen/analysis , Oxygen/metabolism , Partial Pressure , Photosynthesis/genetics , Plants/genetics , Plants/metabolism
10.
J Br Interplanet Soc ; 45(1): 3-12, 1992 Jan.
Article in English | MEDLINE | ID: mdl-11539465

ABSTRACT

A Monte Carlo computer model of extra-solar planetary formation and evolution, which includes the planetary geochemical carbon cycle, is presented. The results of a run of one million galactic disc stars are shown where the aim was to assess the possible abundance of both biocompatible and habitable planets. (Biocompatible planets are defined as worlds where the long-term presence of surface liquid water provides environmental conditions suitable for the origin and evolution of life. Habitable planets are those worlds with more specifically Earthlike conditions). The model gives an estimate of 1 biocompatible planet per 39 stars, with the subset of habitable planets being much rarer at 1 such planet per 413 stars. The nearest biocompatible planet may thus lie approximately 14 LY distant and the nearest habitable planet approximately 31 LY away. If planets form in multiple star systems then the above planet/star ratios may be more than doubled. By applying the results to stars in the solar neighbourhood, it is possible to identify 28 stars at distances of < 22 LY with a non-zero probability of possessing a biocompatible planet.


Subject(s)
Evolution, Planetary , Extraterrestrial Environment , Models, Theoretical , Monte Carlo Method , Planets , Astronomy/methods , Carbon/chemistry , Carbon Dioxide/chemistry , Computer Simulation , Earth, Planet , Evolution, Chemical , Exobiology/methods
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