Infections due to multidrug-resistant bacteria represent a major global health challenge. To combat this problem, new antibiotics are urgently needed and some plant-associated bacteria are a promising source. The rhizobacterium Serratia... more
Infections due to multidrug-resistant bacteria represent a major global health challenge. To combat this problem, new antibiotics are urgently needed and some plant-associated bacteria are a promising source. The rhizobacterium Serratia plymuthica A153 produces several bioactive secondary metabolites, including the anti-oomycete and antifungal haterumalide, oocydin A, and the broad spectrum polyamine antibiotic, zeamine. In this study, we show that A153 produces a second broad spectrum antibiotic, andrimid. Using genome sequencing, comparative genomics and mutagenesis, we defined new genes involved in andrimid (adm) biosynthesis. Both the expression of the adm gene cluster and regulation of andrimid synthesis were investigated. The biosynthetic cluster is operonic and its expression is modulated by various environmental cues, including temperature and carbon source. Analysis of the genome context of the adm operon revealed a gene encoding a predicted LysR-type regulator, AdmX, appar...
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While it is well established that one- and two-component regulatory systems participate in regulating biofilm formation, there also exists evidence suggesting that chemosensory pathways are also involved. However, little information... more
While it is well established that one- and two-component regulatory systems participate in regulating biofilm formation, there also exists evidence suggesting that chemosensory pathways are also involved. However, little information exists about which chemoreceptors and signals modulate this process. Here we report the generation of the complete set of chemoreceptor mutants of Pseudomonas putida KT2440 and the identification of four mutants with significantly altered biofilm phenotypes. These receptors are a WspA homologue of P. aeruginosa, previously identified to control biofilm formation by regulating c-di-GMP levels, and three uncharacterized chemoreceptors. One of these receptors, named McpU, was found to mediate chemotaxis towards different polyamines. The functional annotation of McpU was initiated by high-throughput thermal shift assays of the receptor ligand binding domain (LBD). Isothermal titration calorimetry showed that McpU-LBD specifically binds putrescine, cadaverine...
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The production protocol of many whole cell/virion vaccines involves an inactivation step with β-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL... more
The production protocol of many whole cell/virion vaccines involves an inactivation step with β-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL alkylates nucleotide bases, but its interaction with proteins has not been studied in depth. In the present study we use ellipsometry to analyze the influence of BPL treatment of two H1N1 influenza strains, A/Brisbane/59/2007 and A/New Caledonia/20/1999, which are used for vaccine production on an industrial scale. Analyses were conducted using a mixed lipid monolayer containing ganglioside GM3, which functions as the viral receptor. Our results show that BPL treatment of both strains reduces viral affinity for the mixed monolayer and also diminishes the capacity of viral domains to self-assemble. In another series of experiments, the pH of the subphase was reduced from 7.4 to 5 to provoke the pH-induced conformational change of hemagglutinin, which occu...
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The pea peroxiredoxin homologue PsPrxII F of the Arabidopsis thaliana mitochondrial AtPrxII F was isolated as cDNA and genomic DNA, and characterized in respect to its biochemical and molecular properties. The deduced amino acid sequence... more
The pea peroxiredoxin homologue PsPrxII F of the Arabidopsis thaliana mitochondrial AtPrxII F was isolated as cDNA and genomic DNA, and characterized in respect to its biochemical and molecular properties. The deduced amino acid sequence contains an N-terminal targeting address for mitochondrial import. Mitochondrial location of PsPrxII F was confirmed by immunocytochemistry. The mature enzyme, without the transit peptide, has
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Research Interests: Neuroscience, Psychology, Immunohistochemistry, Gene expression, In Situ Hybridization, and 22 moreHippocampus, Animals, Alcohol Drinking, Male, Astrocyte, Acetylcholine, Basal forebrain, Substantia nigra, Astrocytes, Enzyme, Rats, Time Factors, Degeneration, Autoradiography, Nerve Growth Factor, Trophic Factors, Growth Factor, Somatic Cell Count, Neurosciences, Growth factor receptor, ACH, and Choline Acetyltransferase
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The reasons why some proteins induce a particular type of T helper (Th) response are of fundamental importance but only partially understood. In the present study, amphipatic sequence motifs were identified in N- and C-terminal domains of... more
The reasons why some proteins induce a particular type of T helper (Th) response are of fundamental importance but only partially understood. In the present study, amphipatic sequence motifs were identified in N- and C-terminal domains of Helicobacter pylori (Hp) catalase, which are linked to the induction of Th1 or Th2 immune responses, respectively. Alignment of these motifs with other proteins known to induce either Th1 or Th2 responses has lead to the identification of Th1 and Th2 consensus motifs, termed modulotopes. Their immunomodulatory potential was demonstrated by immunisation experiments using recombinant proteins comprising the C-terminal domain of catalase fused with one or several modulotopes and by co-immunisations of C- or N-terminal catalase domains with peptides containing these motifs. In addition to these in vivo data, in vitro assays using Limulus extracts suggested that modulotopes might interfere with responses triggered by danger signals such as LPS. Th1 and Th2 modulotopes are characterised by a specific hydrophobic/hydrophilic pattern, which might be the structural determinant for their activity. Our data suggest that Th1 and/or Th2 motifs may generally exist on proteins, thus offering the possibility of a rational modulation of the immune response.
Research Interests: Immunology, Immune response, Cytokines, Helicobacter pylori, Hp, and 15 moreMice, Female, Animals, Catalase, Peptides, Vaccination, Protein Conformation, Amino Acid Sequence, Recombinant Protein, Recombinant Proteins, Structure Determination, Antigens, H Pylori, immunoglobulin G, and Molecular Sequence Data
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Chemotaxis is an essential mechanism that enables bacteria to move towards favorable ecological niches. Escherichia coli, the historical model organism for studying chemotaxis, has five well-studied chemoreceptors. However, many bacteria... more
Chemotaxis is an essential mechanism that enables bacteria to move towards favorable ecological niches. Escherichia coli, the historical model organism for studying chemotaxis, has five well-studied chemoreceptors. However, many bacteria with different lifestyle have more chemoreceptors, most of unknown function. Using a high throughput screening approach, we identified a chemoreceptor from Pseudomonas putida KT2440, named McpH, which specifically recognizes purine and its derivatives, adenine, guanine, xanthine, hypoxanthine and uric acid. The latter five compounds form part of the purine degradation pathway, permitting their use as sole nitrogen sources. Isothermal titration calorimetry studies show that these six compounds bind McpH-Ligand Binding Domain (LBD) with very similar affinity. In contrast, non-metabolizable purine derivatives (caffeine, theophylline, theobromine), nucleotides, nucleosides or pyrimidines are unable to bind McpH-LBD. Mutation of mcpH abolished chemotaxis...
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Signal transduction processes are typically initiated by the interaction of signal molecules with sensor domains. The current lack of information on the signal molecules that feed into regulatory circuits forms a major bottleneck that... more
Signal transduction processes are typically initiated by the interaction of signal molecules with sensor domains. The current lack of information on the signal molecules that feed into regulatory circuits forms a major bottleneck that hampers the understanding of regulatory processes. McKellar et al. report a high-throughput approach for the identification of signal molecules, which is based on thermal shift assays of recombinant sensor domains in the absence and presence of compounds from commercially available ligand collections. Initial binding studies with the sensor domain of the PctA chemoreceptor of Pseudomonas aeruginosa showed a close match between thermal shift assay results and microcalorimetric studies reported previously. Using thermal shift assays the authors then identify signals that bind to three chemoreceptors of the kiwifruit pathogen P. syringae pv. Actinidiae NZ-V13. Microcalorimetric binding studies and chemotaxis assays have validated the relevance of these li...
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The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they... more
The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pat...
Research Interests: Protein Folding, Catalysis, Crystallization, Enzyme Inhibitors, Macromolecular X-Ray Crystallography, and 12 moreCatalytic Mechanism, Mycobacterium tuberculosis, Drug Design, Aromatic amino acid, Enzyme, MDR Salmonella typhi, Protein Secondary Structure Prediction, Metabolic pathway, Structure activity Relationship, Shikimic acid, Dimerization, and Molecular Sequence Data
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ABSTRACT Life is based on the specific interaction of molecules and the study of molecular recognition is therefore of fundamental importance. A number of different techniques have been developed to characterise binding events. Amongst... more
ABSTRACT Life is based on the specific interaction of molecules and the study of molecular recognition is therefore of fundamental importance. A number of different techniques have been developed to characterise binding events. Amongst these is Isothermal Titration Calorimetry (ITC) in which heat changes caused by ligand binding is monitored. ITC is becoming increasingly popular, since it has a number of advantages over alternative techniques. ITC does not involve the immobilization or the labelling of ligands and there are little restrictions as to the choice of the buffer system and the analysis temperature. Since heat generation is a generic feature of an interaction, almost all types of ligands can be analysed including small molecules, proteins or ribonucleic acids. However, the major advantages resides in the amount of information obtained in a single experiment which permits the determination of the equilibrium binding constants, the changes in enthalpy (Ho) and entropy (So) as well as information on the binding stoichiometry. In this chapter we provide practical guidelines for the generation of high-quality ITC data giving particular emphasis to trouble shooting. We will illustrate on several examples how ITC can be successfully used to study the binding of hydrocarbons to sensor proteins.
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Research Interests: Molecular Biology, Macromolecular X-Ray Crystallography, Humans, Magnesium, Ligand Binding, and 11 moreHigh Resolution, Active site, Adenylate Kinase, Protein Conformation, Amino Acid Sequence, Protein Binding, Ligands, Binding Site, Biochemistry and cell biology, Adenosine Diphosphate, and Molecular Sequence Data
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A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have... more
A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have essential hyper-reactive arginine residues, and the type II enzymes have an essential tyrosine residue. The essential hyper-reactive Arg-23 of the Streptomyces coelicolor type II enzyme has been replaced by lysine, glutamine, and alanine residues. The mutant enzymes were purified and shown by CD spectroscopy to be structurally similar to the wild-type enzyme. All three mutant enzymes were much less active, for example the kcat of the R23A mutant was 30,000-fold reduced. The mutants all had reduced Km values, indicating stronger substrate binding, which was confirmed by isothermal titration calorimetry experiments. A role for Arg-23 in the stabilization of a carbanion intermediate is proposed. Comparison of the amino acid sequence around the hyper-reactive arginine residues of the two classes of enzymes indicates that there is a conserved structural motif that might reflect a common substrate binding fold at the active center of these two classes of enzyme.
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Research Interests: Biochemistry, Kinetics, Flavonoids, Sequence alignment, Mice, and 12 moreAnimals, Salmonella Typhimurium, P-glycoprotein, Membrane transport proteins, Chalcone, Amino Acid Sequence, Structure activity Relationship, Recombinant Proteins, Binding Site, Biochemistry and cell biology, Adenosine Triphosphate, and Molecular Sequence Data
The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and... more
The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.
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Antibiotic resistance is a widely spread phenomenon. One major mechanism that underlies antibiotic resistance in bacteria is the active extrusion of toxic compounds through the membrane-bound efflux pumps that are often regulated at the... more
Antibiotic resistance is a widely spread phenomenon. One major mechanism that underlies antibiotic resistance in bacteria is the active extrusion of toxic compounds through the membrane-bound efflux pumps that are often regulated at the transcriptional level. TtgR represses the transcription of TtgABC, a key efflux pump in Pseudomonas putida, which is highly resistant to antibiotics, solvents and toxic plant secondary products. Previously we showed that TtgR is the only reported repressor that binds to different classes of natural antimicrobial compounds, which are also extruded by the efflux pump. We report here five high-resolution crystal structures of TtgR from the solvent-tolerant strain DOT-T1E, including TtgR in complex with common antibiotics and plant secondary metabolites. We provide structural basis for the unique ligand binding properties of TtgR. We identify two distinct and overlapping ligand binding sites; the first one is broader and consists of mainly hydrophobic residues, whereas the second one is deeper and contains more polar residues including Arg176, a unique residue present in the DOT-T1E strain but not in other Pseudomonas strains. Phloretin, a plant antimicrobial, can bind to both binding sites with distinct binding affinities and stoichiometries. Results on ligand binding properties of native and mutant TtgR proteins using isothermal titration calorimetry confirm the binding affinities and stoichiometries, and suggest a potential positive cooperativity between the two binding sites. The importance of Arg176 in phloretin binding was further confirmed by the reduced ability of phloretin in releasing the mutant TtgR from bound DNA compared to the native protein. The results presented here highlight the importance and versatility of regulatory systems in bacterial antibiotic resistance and open up new avenues for novel antimicrobial development.
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TtgR is the specific transcriptional repressor of the TtgABC efflux pump. TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype of... more
TtgR is the specific transcriptional repressor of the TtgABC efflux pump. TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype of Pseudomonas putida DOT-T1E. TtgR binds to a pseudo-palindromic site that overlaps the ttgR/ttgA promoters. Dimethylsulfate footprint assays reveal a close interaction between TtgR and the central region of this operator. The results of analytical ultracentrifugation demonstrate that TtgR forms stable dimers in solution, and that two dimers bind to the operator. Microcalorimetric analysis of the binding of the two TtgR dimers to the cognate operator showed biphasic behavior, and an interaction model was developed for the cooperative binding of two TtgR dimers to their target operators. The binding of the two TtgR dimers to the operator was characterized by a Hill coefficient of 1.63+/-0.13 (k(D)=18.2(+/-6.3) microM, k(D)(')=0.91(+/-0.49) microM), indi...
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Research Interests: Thermodynamics, Molecular Biology, Molecular, Protein-DNA interaction, Mutation, and 14 morePhosphorylation, Pseudomonas putida, Amino Acid Profile, Isothermal Titration Calorimetry, Molecular weight, Base Sequence, Protein Binding, Binding Site, DNA-footprinting, Transcription Start Site, Full Length Movies, Biochemistry and cell biology, In Vitro Transcription, and Binding affinity
The structure of the type II DHQase from Streptomyces coelicolor has been solved and refined to high resolution in complexes with a number of ligands, including dehydroshikimate and a rationally designed transition state analogue,... more
The structure of the type II DHQase from Streptomyces coelicolor has been solved and refined to high resolution in complexes with a number of ligands, including dehydroshikimate and a rationally designed transition state analogue, 2,3-anhydro-quinic acid. These structures define the active site of the enzyme and the role of key amino acid residues and provide snap shots of the catalytic cycle. The resolution of the flexible lid domain (residues 21-31) shows that the invariant residues Arg23 and Tyr28 close over the active site cleft. The tyrosine acts as the base in the initial proton abstraction, and evidence is provided that the reaction proceeds via an enol intermediate. The active site of the structure of DHQase in complex with the transition state analog also includes molecules of tartrate and glycerol, which provide a basis for further inhibitor design.
Research Interests: Structure, Protein Folding, Macromolecular X-Ray Crystallography, Biological Sciences, Streptomyces, and 13 moreSequence alignment, Animals, Enzyme, Enzyme Mechanism, CHEMICAL SCIENCES, Rational drug design, High Resolution, Active site, Amino Acid Profile, Amino Acid Sequence, Protein Quaternary Structure, Shikimic acid, and Molecular Structure
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A gene coding for a small heat shock protein homolog from the hyperthermophilic methanogenic ArchaeonMethanococcus jannaschiiwas cloned. This gene was overexpressed inEscherichia coliharboring rare codon tRNAs and its protein purified and... more
A gene coding for a small heat shock protein homolog from the hyperthermophilic methanogenic ArchaeonMethanococcus jannaschiiwas cloned. This gene was overexpressed inEscherichia coliharboring rare codon tRNAs and its protein purified and crystallized. Crystals ...