Publications

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the hyperthermophilic nature of the metallo-oxidase from aquifex aeolicus.the stability of the aquifex aeolicus multicopper oxidase (mcoa) was studied by spectroscopy, calorimetry and chromatography to understand its thermophilic nature. the enzyme is hyperthermostable as deconvolution of the differential scanning calorimetry trace shows that thermal unfolding is characterized by temperature values at the mid-point of 105, 110 and 114 degrees c. chemical denaturation revealed however a very low stability at room temperature (2.8 kcal/mol) because copper bleaching/depl ...200918930169
structure and regulatory mechanism of aquifex aeolicus ntrc4: variability and evolution in bacterial transcriptional regulation.genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. comparative studies provide insights into the functional consequences of specific changes. here we present structural and biochemical studies of ntrc4, a sigma-54 activator from aquifex aeolicus, and compare it with ntrc1 (a paralog) and ntrc (a homolog from salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may hav ...200818955063
monitoring assembly of ribonucleoprotein complexes by isothermal titration calorimetry.isothermal titration calorimetry (itc) is a useful technique to study rna-protein interactions as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. this chapter presents a general procedure for studying rna-protein interactions using itc and gives specific examples for monitoring the binding of caenorhabditis elegans gld-1 star domain to tge rna and the binding of aquifex aeolicus s6:s18 ribosomal protein heterodim ...200818982287
methionine uptake in corynebacterium glutamicum by metqni and by metps, a novel methionine and alanine importer of the nss neurotransmitter transporter family.the soil bacterium corynebacterium glutamicum is a model organism in amino acid biotechnology. here we present the identification of two different l-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. the primary carrier metqni is a high affinity abc-type transporter specific for l-methionine. its expression is under the control of the transcription factor mcbr, the global regulator of sulfur metabolism in c. glutamicum. besides metqni, a n ...200818991398
heterologous production, isolation, characterization and crystallization of a soluble fragment of the nadh:ubiquinone oxidoreductase (complex i) from aquifex aeolicus.the proton-pumping nadh:ubiquinone oxidoreductase (complex i) is the first enzyme complex of the respiratory chains in many bacteria and most eukaryotes. it is the least understood of all, due to its enormous size and unique energy conversion mechanism. the bacterial complex is in general made up of 14 different subunits named nuoa-n. subunits nuoe, -f, and -g comprise the electron input part of the complex. we have cloned these genes from the hyperthermophilic bacterium aquifex aeolicus and exp ...200819006332
structural synergy and molecular crosstalk between bacterial helicase loaders and replication initiators.the loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. in cells, dedicated aaa+ atpases regulate loading, however, the mechanism by which these factors recruit and deposit helicases has remained unclear. to better understand this process, we determined the structure of the atpase region of the bacterial helicase loader dnac from aquifex aeolicus to 2.7 a resolution. the structure shows that dnac is a close paralog of the bacterial replication ...200819013274
structure of (e)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the non-mevalonate pathway.molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the 2-c-methyl-d-erythritol-4-phosphate (mep) pathway. the mep pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, plasmodium falciparum) as well as by plants, but is not present in animals. the terminal reaction of the mep pathway is catalyzed by (e)-4-hydroxy-3-methyl-but-2-enyl diphosphate (hmbpp) reductase (lytb), an enzyme that converts hm ...200819035630
accounting for horizontal gene transfers explains conflicting hypotheses regarding the position of aquificales in the phylogeny of bacteria.despite a large agreement between ribosomal rna and concatenated protein phylogenies, the phylogenetic tree of the bacterial domain remains uncertain in its deepest nodes. for instance, the position of the hyperthermophilic aquificales is debated, as their commonly observed position close to thermotogales may proceed from horizontal gene transfers, long branch attraction or compositional biases, and may not represent vertical descent. indeed, another view, based on the analysis of rare genomic c ...200818834516
first characterisation of the active oligomer form of sulfur oxygenase reductase from the bacterium aquifex aeolicus.sulfur oxygenase reductase (sor) enzyme is responsible for the initial oxidation step of elemental sulfur in archaea. curiously, aquifex aeolicus, a hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium, has the sor-encoding gene in its genome. we showed, for the first time the presence of the sor enzyme in a. aeolicus, its gene was cloned and recombinantly expressed in escherichia coli and the protein was purified and characterised. it is a 16 homo-oligomer of approximately 600 ...200818060346
rna processing in aquifex aeolicus involves rnase e/g and an rnase p-like activity.to assess the evolutionary conservation of rna processing pathways in aquifex aeolicus, we characterized the products of rrna and trna processing that originated from polycistronic transcripts encoded by the a. aeolicus tufa2 and rrna operons. we found that, similar to its escherichia coli counterpart, a. aeolicus rnase e/g is involved in rrna processing and maturation of trnas, thus indicating that rna processing pathways in e. coli and a. aeolicus include common steps and some of them are depe ...200818070593
discrimination of mesophilic and thermophilic proteins using machine learning algorithms.discriminating thermophilic proteins from their mesophilic counterparts is a challenging task and it would help to design stable proteins. in this work, we have systematically analyzed the amino acid compositions of 3075 mesophilic and 1609 thermophilic proteins belonging to 9 and 15 families, respectively. we found that the charged residues lys, arg, and glu as well as the hydrophobic residues, val and ile have higher occurrence in thermophiles than mesophiles. further, we have analyzed the per ...200817876820
synthesis and characterization of cytidine derivatives that inhibit the kinase ispe of the non-mevalonate pathway for isoprenoid biosynthesis.the enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. this pathway is used exclusively by the corresponding pathogens, but not by humans. a series of water-soluble, cytidine-based inhibitors that were originally designed for the fourth enzyme in the pathway, ispd, were shown to inhibit the subsequent enzyme, the kinase ispe (from escherichia coli). the binding mode of the inhibitors was ve ...200818033714
the c-terminal region of thermophilic trna (m7g46) methyltransferase (trmb) stabilizes the dimer structure and enhances fidelity of methylation.transfer rna (m(7)g46) methyltransferase catalyzes methyl-transfer from s-adenosyl-l-methionine to n(7) atom of the semi-conserved g46 base in trna. aquifex aeolicus is a hyper thermophilic eubacterium that grows at close to 95 degrees c. a. aeolicus trna (m(7)g46) methyltransferase [trmb] has an elongated c-terminal region as compared with mesophilic counterparts. in this study, the authors focused on the functions of this c-terminal region. analytic gel filtration chromatography and amino acid ...200818076049
structure of the ppx/gppa phosphatase from aquifex aeolicus in complex with the alarmone ppgpp.the crystal structure of the prototype exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family member from aquifex aeolicus in complex with the intracellular second messenger guanosine tetraphosphate was determined at 2.7-a resolution. the hydrolytic base is identified as e119. the dual specificity established for the escherichia coli homolog is shown to be compatible with a common active site for guanosine pentaphosphate and polyphosphate hydrolysis. distinct and different d ...200818155044
functional role of conserved residues in the characteristic secretion ntpase motifs of the pseudomonas aeruginosa type iv pilus motor proteins pilb, pilt and pilu.type iv pili are retractable protein fibres used by many bacterial pathogens for adherence, twitching motility, biofilm development and host colonization. in pseudomonas aeruginosa, pilb and pilt are bipolar proteins belonging to the secretion ntpase superfamily, and power pilus extension and retraction, respectively, while the unipolar pilt paralogue pilu supports pilus retraction in an unknown manner. assay of purified 6xhis-tagged pilb, pilt and pilu from p. aeruginosa showed that all three p ...200818174131
structural biophysics of the nusb:nuse antitermination complex.in prokaryotic transcription regulation, several host factors form a complex with rna polymerase and the nascent mrna. as part of a process known as antitermination, two of these host factors, nusb and nuse, bind to form a heterodimer, which interacts with a specific boxa site on the rna. the nusb/nuse/boxa rna ternary complex interacts with the rna polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. the nusb protein also binds boxa rna ...200818177898
5'-end maturation of trna in aquifex aeolicus.5'-end maturation of trna primary transcripts is thought to be ubiquitously catalyzed by ribonuclease p (rnase p), a ribonucleoprotein enzyme in the vast majority of organisms and organelles. in the hyperthermophilic bacterium aquifex aeolicus, neither a gene for the rna nor the protein component of bacterial rnase p has been identified in its sequenced genome. here, we demonstrate the presence of an rnase p-like activity in cell lysates of a. aeolicus. detection of activity was sensitive to the ...200818208351
the sole serine/threonine protein kinase and its cognate phosphatase from aquifex aeolicus targets pyrimidine biosynthesis.serine/threonine kinases participate in complex, interacting signaling pathways in eukaryotes, prokaryotes, and archae. while most organisms contain many different kinases, the extreme hyperthermophile, aquifex aeolicus encodes a single hypothetical ser/thr kinase. a gene homologous to eukaryotic protein phosphatases overlaps the kinase gene by a single base pair. the putative kinase, aastpk and phosphatase, aappm, were cloned and expressed in e. coli, purified to homogeneity and found to be fun ...200818270660
mechanism and inhibition of lpxc: an essential zinc-dependent deacetylase of bacterial lipid a synthesis.multi-drug resistant (mdr), pathogenic gram-negative bacteria pose a serious health threat, and novel antibiotic targets must be identified to combat mdr infections. one promising target is the zinc-dependent metalloamidase udp-3-o-(r-3-hydroxymyristoyl)-n-acetylglucosamine deacetylase (lpxc), which catalyzes the committed step of lipid a (endotoxin) biosynthesis. lpxc is an essential, single copy gene that is conserved in virtually all gram-negative bacteria. lpxc structures, revealed by nmr an ...200818289052
structural studies of thiamin monophosphate kinase in complex with substrates and products.thiamin monophosphate kinase (thil) catalyzes the atp-dependent phosphorylation of thiamin monophosphate (tmp) to form thiamin pyrophosphate (tpp), the active form of vitamin b 1. thil is a member of a small atp binding superfamily that also includes the purine biosynthetic enzymes, purm and purl, nife hydrogenase maturation protein, hype, and selenophosphate synthase, seld. the latter four enzymes are believed to utilize phosphorylated intermediates during catalysis. to understand the mechanism ...200818311927
binding of serotonin to the human serotonin transporter. molecular modeling and experimental validation.molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-ht) to the human serotonin transporter (hsert). homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from aquifex aeolicus, as the template and three slightly different sequence alignments. induced fit docking of 5-ht into hsert homology models resulted in two different binding modes. both show ...200818314975
comparative molecular field analysis using selectivity fields reveals residues in the third transmembrane helix of the serotonin transporter associated with substrate and antagonist recognition.the human serotonin transporter (hsert) regulates the spatial and temporal actions of serotonin (5-ht) neurotransmission by removing 5-ht from the synapse. previous studies have identified residues in the third transmembrane helix (tmh) that may be important for substrate translocation or antagonist recognition. we identified hsert residues in tmh iii that are divergent from drosophila sert and used species-scanning mutagenesis to generate reciprocal mutants. transport inhibition assays suggest ...200818354055
recognition of trnaleu by aquifex aeolicus leucyl-trna synthetase during the aminoacylation and editing steps.recognition of trna by the cognate aminoacyl-trna synthetase during translation is crucial to ensure the correct expression of the genetic code. to understand trna(leu) recognition sets and their evolution, the recognition of trna(leu) by the leucyl-trna synthetase (leurs) from the primitive hyperthermophilic bacterium aquifex aeolicus was studied by rna probing and mutagenesis. the results show that the base a73; the core structure of trna formed by the tertiary interactions u8-a14, g18-u55 and ...200818367476
membrane structure of ctra3, a copper-transporting p-type-atpase from aquifex aeolicus.we have produced and characterized two new copper-transporting atpases, ctra2 and ctra3 from aquifex aeolicus, that belong to the family of heavy metal ion-transporting p(ib)-type atpases. ctra2 has a cpc metal-binding sequence in tm6 and a cxxc metal-binding n-terminal domain, while ctra3 has a cph metal-binding motif in tm6 and a histidine-rich n-terminal metal-binding domain. we have cloned both copper pumps, expressed them in escherichia coli and characterized them functionally. ctra2 is act ...200818374940
characterization of aquifex aeolicus 4-diphosphocytidyl-2c-methyl-d-erythritol kinase - ligand recognition in a template for antimicrobial drug discovery.4-diphosphocytidyl-2c-methyl-d-erythritol kinase (ispe) catalyses the atp-dependent conversion of 4-diphosphocytidyl-2c-methyl-d-erythritol (cdpme) to 4-diphosphocytidyl-2c-methyl-d-erythritol 2-phosphate with the release of adp. this reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. we set out to characterize the biochem ...200818422643
variation of average g values and effective exchange coupling constants among [2fe-2s] clusters: a density functional theory study of the impact of localization (trapping forces) versus delocalization (double-exchange) as competing factors.a phenomenological model aimed at rationalizing variations in both average g-tensor values (gav identical with 1/3sigmaigi ) and effective exchange coupling constants jeff (defined as two-thirds of the energy difference between the s = 3/2 and s = 1/2 spin states) has been derived in order to describe the great variety of magnetic properties exhibited by reduced [2fe-2s] clusters in proteins. the key quantity in the present analysis is the ratio delta e/b computed from two competing terms. delta ...200818491857
dynamics of rhodobacter capsulatus [2fe-2s] ferredoxin vi and aquifex aeolicus ferredoxin 5 via nuclear resonance vibrational spectroscopy (nrvs) and resonance raman spectroscopy.we have used (57)fe nuclear resonance vibrational spectroscopy (nrvs) to study the fe(2)s(2)(cys)(4) sites in oxidized and reduced [2fe-2s] ferredoxins from rhodobacter capsulatus (rc fdvi) and aquifex aeolicus (aa fd5). in the oxidized forms, nearly identical nrvs patterns are observed, with strong bands from fe-s stretching modes peaking around 335 cm(-1), and additional features observed as high as the b(2u) mode at approximately 421 cm(-1). both forms of rc fdvi have also been investigated b ...200818512953
structure of an n-terminally truncated selenophosphate synthetase from aquifex aeolicus.selenophosphate synthetase (sps) catalyzes the activation of selenide with atp to synthesize selenophosphate, the reactive selenium donor for biosyntheses of both the 21st amino acid selenocysteine and 2-selenouridine nucleotides in trna anticodons. the crystal structure of an n-terminally (25 residues) truncated fragment of sps (sps-deltan) from aquifex aeolicus has been determined at 2.0 a resolution. the structure revealed sps to be a two-domain alpha/beta protein, with domain folds that are ...200818540050
location and flexibility of the unique c-terminal tail of aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques.co-chaperonin protein 10 (cpn10, groes in escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (groel in e. coli). the cpn10 from the hyperthermophilic, ancient bacterium aquifex aeolicus (aacpn10) has a 25-residue c-terminal extension in each monomer not found in any other cpn10 protein. earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. without the tail, however, the heptamers (a ...200818588898
helix xi contributes to the entrance of the serotonin transporter permeation pathway.the sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (mpp(+)). previous studies implicated residues in transmembrane helix (tmh) xi of dat as important sites for mpp(+) transport. we examined the importance of tmh xi residues f551 and f556 for mpp(+) translocation by human sert. mutations at hsert f556, but not f551, reduced both 5-ht and mpp(+) transport compared to wild type. h ...200818628241
inhibitors of the kinase ispe: structure-activity relationships and co-crystal structure analysis.enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are therapeutic targets for the treatment of important infectious diseases. whereas this pathway is absent in humans, it is used by plants, many eubacteria and apicomplexan protozoa, including major human pathogens such as plasmodium falciparum and mycobacterium tuberculosis. herein, we report on the design, preparation and biological evaluation of a new series of ligands for ispe protein, a kinase from this pathway. these inhibit ...200818633530
structural analysis of the contacts anchoring moenomycin to peptidoglycan glycosyltransferases and implications for antibiotic design.peptidoglycan glycosyltransferases (pgts), enzymes that catalyze the formation of the glycan chains of the bacterial cell wall, have tremendous potential as antibiotic targets. the moenomycins, a potent family of natural product antibiotics, are the only known active site inhibitors of the pgts and serve as blueprints for the structure-based design of new antibacterials. a 2.8 a structure of a staphylococcus aureus pgt with moenomycin a bound in the active site appeared recently, potentially pro ...200818642800
the highest affinity binding site of small protein b on transfer messenger rna is outside the trna domain.eubacterial ribosomes stalled on defective mrnas are released through a mechanism referred to as trans-translation, depending on the coordinated actions of small protein b (smpb) and transfer messenger rna (tmrna). a series of tmrna variants with deletions in each structural domain were produced. their structures were monitored by enzymatic and chemical probes in vitro, in the presence and absence of smpb. dissociation constants between these rnas and smpb from aquifex aeolicus were derived by s ...200818648069
stabilization of trna (mg37) methyltransferase [trmd] from aquifex aeolicus by an intersubunit disulfide bond formation.recombinant aquifex aeolicus trmd protein has a cys20-cys20 disulfide bond between its two subunits. this was demonstrated by sds-polyacrylamide gel analysis of wild-type enzyme and c20s mutant protein (in which the cys20 residue is substituted by serine), in the absence or presence of various concentrations of dithiothreitol. analytical gel-filtration chromatography revealed that the c20s mutant protein forms a dimer structure even though it is missing the disulfide bond. western blotting analy ...200818651851
2,5-diamino-6-ribitylamino-4(3h)-pyrimidinone 5'-phosphate synthases of fungi and archaea.the pathway of riboflavin (vitamin b2) biosynthesis is significantly different in archaea, eubacteria, fungi and plants. specifically, the first committed intermediate, 2,5-diamino-6-ribosylamino-4(3h)-pyrimidinone 5'-phosphate, can either undergo hydrolytic cleavage of the position 2 amino group by a deaminase (in plants and most eubacteria) or reduction of the ribose side chain by a reductase (in fungi and archaea). we compare 2,5-diamino-6-ribitylamino-4(3h)-pyrimidinone 5'-phosphate synthase ...200818671734
hyperthermophilic aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines.the placement of the extreme thermophile aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. we investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically dnag primase. although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for a. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. to determi ...200818684998
the asymmetric trimeric architecture of [2fe-2s] iscu: implications for its scaffolding during iron-sulfur cluster biosynthesis.iscu is a key component of the isc machinery and is involved in the biogenesis of iron-sulfur (fe-s) proteins. iscu serves as a scaffold for assembly of a nascent fe-s cluster prior to its delivery to an apo protein. here, we report the first crystal structure of iscu with a bound [2fe-2s] cluster from the hyperthermophilic bacterium aquifex aeolicus, determined at a resolution of 2.3 a, using multiwavelength anomalous diffraction of the cluster. the holo iscu formed a novel asymmetric trimer th ...200818723024
residue ionization in lpxc directly observed by 67zn nmr spectroscopy.the ph dependence of the solid-state (67)zn nmr lineshapes has been measured for both the wild type (wt) and the h265a mutant of aquifex aeolicus lpxc, each in the absence of substrate (resting state). the (67)zn nmr spectrum of wt lpxc at ph 6 (prepared at 0 degrees c) contains two overlapping quadrupole lineshapes with c q values of 10 and 12.9 mhz, while the spectrum measured for the sample prepared at a ph near 9 (at 0 degrees c) is dominated by the appearance of a third species with a c q o ...200818761443
new approaches to understanding double-stranded rna processing by ribonuclease iii purification and assays of homodimeric and heterodimeric forms of rnase iii from bacterial extremophiles and mesophiles.ribonuclease iii (rnase iii) is a double-stranded (ds)-rna-specific endonuclease that plays essential roles in the maturation and decay of coding and noncoding rnas. bacterial rnases iii are structurally the simplest members of the rnase iii family, which includes the eukaryotic orthologs dicer and drosha. high-resolution crystal structures of rnase iii of the hyperthermophilic bacteria aquifex aeolicus and thermotoga maritima are available. a. aeolicus rnase iii also has been cocrystallized wit ...200819161841
the slc6/nss family members kaat1 and caatch1 have weak chloride dependence.kaat1 and caatch1 are amino acid transporters cloned from the intestine of the lepidoptera manduca sexta. they are members of the slc6/nss family, which groups membrane proteins that use na(+), k(+), and cl⁻ gradients for the coupled transport of amines and amino acids. the report of the atomic-resolution x-ray crystal structure of the eubacterium aquifex aeolicus leucine transporter (aaleut) has contributed significantly to understanding of the structure-function relationship in nss proteins. t ...200819066444
bound to be different: neurotransmitter transporters meet their bacterial cousins.the neurotransmitter transporters belonging to the solute carrier 6 (slc6) family, including the gamma-aminobutyric acid (gat), norepinephrine (net), serotonin (sert) and dopamine (dat) transporters are extremely important drug targets of great clinical relevance. these na+, cl(-)-dependent transporters primarily function following neurotransmission to reset neuronal signaling by transporting neurotransmitter out of the synapse and back into the pre-synaptic neuron. recent studies have tracked d ...200718199851
domain motions of argonaute, the catalytic engine of rna interference.the argonaute protein is the core component of the rna-induced silencing complex, playing the central role of cleaving the mrna target. visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of argonaute that might occur during rna interference. we have taken the next step by performing an all-atom normal mode analysis of the pyrococcus furiosus and aquifex aeolicus argonaute crystal structures, allowing us to quantitatively assess the fe ...200718053142
structural insights into the conformational variability of ftsz.ftsz is a prokaryotic homologue of the eukaryotic cytoskeletal protein tubulin and plays a central role in prokaryotic cell division. both ftsz and tubulin are known to pass through cycles of polymerization and depolymerization, but the structural mechanisms underlying this cycle remain to be determined. comparison of tubulin structures obtained in different states has led to a model in which the tubulin monomer undergoes a conformational switch between a "straight" form found in the walls of mi ...200717900614
structural basis of the initial binding of trna(ile) lysidine synthetase tils with atp and l-lysine.in the bacterial genetic-code system, the codon aua is decoded as isoleucine by trna(ile)(2) with the lysidine residue at the wobble position. lysidine is derived from cytidine, with atp and l-lysine, by trna(ile) lysidine synthetase (tils), which is an n-type atp pyrophosphatase. in this study, we determined the crystal structure of aquifex aeolicus tils, complexed with atp, mg2+, and l-lysine, at 2.5 a resolution. the presence of the tils-specific subdomain causes the active site to have two s ...200718073113
folding of s6 structures with divergent amino acid composition: pathway flexibility within partly overlapping foldons.studies of circular permutants have demonstrated that the folding reaction of s6 from thermus thermophilus (s6(t)) is malleable and responds in an ordered manner to changes of the sequence separation between interacting residues: the s6(t) permutants retain a common nucleation pattern in the form of a two-strand-helix motif that can be recruited from different parts of the structure. to further test the robustness of the two-strand-helix nucleus we have here determined the crystal structure and ...200717056063
crystal structure of the bifunctional atp sulfurylase-aps kinase from the chemolithotrophic thermophile aquifex aeolicus.the thermophilic chemolithotroph, aquifex aeolicus, expresses a gene product that exhibits both atp sulfurylase and adenosine-5'-phosphosulfate (aps) kinase activities. these enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. the domain arrangement in the aquifex enzyme is reminiscent of the fungal atp sulfurylase, which contains a c-terminal domain that is homologous to aps kinase yet displays no kinase activity. rather, in the fungal enzyme, the motif ...200717095009
a present-day aminoacyl-trna synthetase with ancestral editing properties.leucyl-, isoleucyl-, and valyl-trna synthetases form a subgroup of related aminoacyl-trna synthetases that attach similar amino acids to their cognate trnas. to prevent amino acid misincorporation during translation, these enzymes also hydrolyze mischarged trnas through a post-transfer editing mechanism. here we show that leurs from the deep-branching bacterium aquifex aeolicus edits the complete set of aminoacylated trnas generated by the three enzymes: ile-trna(ile), val-trna(ile), val-trna(va ...200717095543
structure of aquifex aeolicus argonaute highlights conformational flexibility of the paz domain as a potential regulator of rna-induced silencing complex function.gene silencing mediated by rna interference requires the sequence-specific recognition of target mrna by the endonuclease argonaute, the primary enzymatic component of the rna-induced silencing complex. we report the crystal structure of aquifex aeolicus argonaute, refined at 3.2a resolution. relative to recent argonaute structures, a 24 degrees reorientation of the paz domain in our structure opens a basic cleft between the n-terminal and paz domains, exposing the guide strand binding pocket of ...200717130125
reengineering cca-adding enzymes to function as (u,g)- or dcdcda-adding enzymes or poly(c,a) and poly(u,g) polymerases.cca-adding enzymes build and repair the 3'-terminal cca sequence of trna. these unusual rna polymerases use either a ribonucleoprotein template (class i) or pure protein template (class ii) to form mock base pairs with the watson-crick edges of incoming ctp and atp. guided by the class ii bacillus stearothermophilus cca-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. we were able to transform the cc ...200717179213
localization of cocaine analog [125i]rti 82 irreversible binding to transmembrane domain 6 of the dopamine transporter.the site of cocaine binding on the dopamine transporter (dat) was investigated using the photoactivatable irreversible cocaine analog [125i]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125i]rti 82). the incorporation site of this compound was mapped to transmembrane domains (tms) 4-6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (cnbr), which hydrolyzes proteins on the c-terminal side of me ...200717255098
crystallization and x-ray diffraction analysis of the beta-ketoacyl-acyl carrier protein reductase fabg from aquifex aeolicus vf5.the gene product of fabg from aquifex aeolicus has been heterologously expressed in escherichia coli. purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. diffraction data were collected to a maximum resolution of 1.8 a and the initial phases were determined by molecular replacement. the a. aeolicus fabg protein is a putative beta-ketoacyl-acyl carrier protein reductase. structure-function studies of this protein are ...200717277451
inhibition of lipid a biosynthesis as the primary mechanism of chir-090 antibiotic activity in escherichia coli.the deacetylation of udp-3-o-[(r)-3-hydroxymyristoyl]-n-acetylglucosamine (udp-3-o-acyl-glcnac) by lpxc is the committed reaction of lipid a biosynthesis. chir-090, a novel n-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the lpxc deacetylase from the hyperthermophile aquifex aeolicus, and it has excellent antibiotic activity against pseudomonas aeruginosa and escherichia coli, as judged by disk diffusion assays. we now report that chir-090 is also a two-step sl ...200717335290
crystal structures of the pilus retraction motor pilt suggest large domain movements and subunit cooperation drive motility.pilt is a hexameric atpase required for bacterial type iv pilus retraction and surface motility. crystal structures of adp- and atp-bound aquifex aeolicus pilt at 2.8 and 3.2 a resolution show n-terminal pas-like and c-terminal reca-like atpase domains followed by a set of short c-terminal helices. the hexamer is formed by extensive polar subunit interactions between the atpase core of one monomer and the n-terminal domain of the next. an additional structure captures a nonsymmetric pilt hexamer ...200717355871
crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis.peptidoglycan is an essential polymer that forms a protective shell around bacterial cell membranes. peptidoglycan biosynthesis is the target of many clinically used antibiotics, including the beta-lactams, imipenems, cephalosporins, and glycopeptides. resistance to these and other antibiotics has prompted interest in an atomic-level understanding of the enzymes that make peptidoglycan. representative structures have been reported for most of the enzymes in the pathway. until now, however, there ...200717360321
structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-d-manno-octulosonate 8-phosphate synthases.there are two classes of kdo8p synthases characterized respectively by the presence or absence of a metal in the active site. the nonmetallo kdo8ps from escherichia coli and the metallo kdo8ps from aquifex aeolicus are the best characterized members of each class. all amino acid residues that make important contacts with the substrates are conserved in both enzymes with the exception of pro-10, cys-11, ser-235, and gln-237 of the a. aeolicus enzyme, which correspond respectively to met-25, asn-2 ...200717381075
homology modeling of the serotonin transporter: insights into the primary escitalopram-binding site.the serotonin transporter (sert) is one of the neurotransmitter transporters that plays a critical role in the regulation of endogenous amine concentrations and therefore is an important target for therapeutic agents affecting the central nervous system. the recently published, high resolution x-ray structure of the closely related amino acid transporter, aquifex aeolicus leucine transporter (leut), provides an opportunity to develop a three-dimensional model of the structure of sert. we present ...200717405130
mechanism and putative structure of b(0)-like neutral amino acid transporters.the na(+)-dependent transport of neutral amino acids in epithelial cells and neurons is mediated by b(0)-type neutral amino acid transporters. two b(0)-type amino acid transporters have been identified in the neurotransmitter transporter family slc6, namely b(0)at1 (slc6a19) and b(0)at2 (slc6a15). in contrast to other members of this family, b(0)-like transporters are chloride-independent. b(0)at1 and b(0)at2 preferentially bind the substrate prior to the na(+)-ion. the na(+)-concentration affec ...200717417702
molecular dynamics simulations of na+/cl(-)-dependent neurotransmitter transporters in a membrane-aqueous system.we have performed molecular dynamics simulations of a homology model of the human serotonin transporter (hsert) in a membrane environment and in complex with either the natural substrate 5-ht or the selective serotonin reuptake inhibitor escitalopram. we have also included a transporter homologue, the aquifex aeolicus leucine transporter (leut), in our study to evaluate the applicability of a simple and computationally attractive membrane system. fluctuations in leut extracted from simulations a ...200717436258
atp ground- and transition states of bacterial enhancer binding aaa+ atpases support complex formation with their target protein, sigma54.transcription initiation by the sigma54 form of bacterial rna polymerase requires hydrolysis of atp by an enhancer binding protein (ebp). we present sas-based solution structures of the atpase domain of the ebp ntrc1 from aquifex aeolicus in different nucleotide states. structures of apo protein and that bound to amppnp or adp-bef(x) (ground-state mimics), adp-alf(x) (a transition-state mimic), or adp (product) show substantial changes in the position of the gaftga loops that contact polymerase, ...200717437715
a robust metallo-oxidase from the hyperthermophilic bacterium aquifex aeolicus.the gene, aquifex aeolicus aac07157.1, encoding a multicopper oxidase (mcoa) and localized in the genome as part of a putative copper-resistance determinant, has been cloned, over-expressed in escherichia coli and the recombinant enzyme purified to homogeneity. the purified enzyme shows spectroscopic and biochemical characteristics typical of the well-characterized multicopper oxidase family of enzymes. mcoa presents higher specificity (k(cat)/k(m)) for cuprous and ferrous ions than for aromatic ...200717451433
bifunctional ctp:inositol-1-phosphate cytidylyltransferase/cdp-inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper)thermophiles.the pathway for the synthesis of di-myo-inositol-phosphate (dip) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (nmr) (n. borges, l. g. gonçalves, m. v. rodrigues, f. siopa, r. ventura, c. maycock, p. lamosa, and h. santos, j. bacteriol. 188:8128-8135, 2006). here, a genomic approach was used to identify the genes involved in the synthesis of dip. ...200717526717
structure of o67745_aquae, a hypothetical protein from aquifex aeolicus.using single-wavelength anomalous dispersion data obtained from a gold-derivatized crystal, the x-ray crystal structure of the protein 067745_aquae from the prokaryotic organism aquifex aeolicus has been determined to a resolution of 2.0 a. amino-acid residues 1-371 of the 44 kda protein were identified by pfam as an hd domain and a member of the metal-dependent phosphohydrolase superfamily (accession no. pf01966). although three families from this large and diverse group of enzymatic proteins a ...200717565173
protein aq_1862 from the hyperthermophilic bacterium aquifex aeolicus is a porin and contains two conductance pathways of different selectivity.the "hypothetical protein" aq_1862 was isolated from the membrane fraction of aquifex aeolicus and identified as the major porin. in experiments with one conducting unit (molecule) a conductance of 1.4 ns was observed in 0.1 m kcl at ph 7.5. this stable (basic) conductance was superimposed by conductance fluctuations of approximately 0.25 ns. because both events were always observed simultaneously, it is suggested that they are caused by the same molecular entity. nonetheless they show very diff ...200717586565
structural and functional analysis of two glutamate racemase isozymes from bacillus anthracis and implications for inhibitor design.glutamate racemase (race) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen bacillus anthracis. race enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. we have cloned, expressed and purified the two glutamate racemase isozymes, race1 an ...200717610893
phylogenetic diversity and the structural basis of substrate specificity in the beta/alpha-barrel fold basic amino acid decarboxylases.the beta/alpha-barrel fold type basic amino acid decarboxylases include eukaryotic ornithine decarboxylases (odc) and bacterial and plant enzymes with activity on l-arginine and meso-diaminopimelate. these enzymes catalyze essential steps in polyamine and lysine biosynthesis. phylogenetic analysis suggests that diverse bacterial species also contain odc-like enzymes from this fold type. however, in comparison with the eukaryotic odcs, amino acid differences were identified in the sequence of the ...200717626020
structure of the biotin carboxylase domain of pyruvate carboxylase from bacillus thermodenitrificans.the biotin carboxylase (bc) domain of pyruvate carboxylase (pc) from bacillus thermodenitrificans (bc-bpc) was crystallized in an orthorhombic form (space group p2(1)2(1)2(1)), with unit-cell parameters a = 79.6, b = 116.0, c = 115.7 a. two bc protomers are contained in the asymmetric unit. diffraction data were collected at 100 k and the crystal structure was solved by the molecular-replacement method and refined against reflections in the 20.0-2.4 a resolution range, giving an r factor of 0.23 ...200717642515
structural and biochemical analyses of shikimate dehydrogenase aroe from aquifex aeolicus: implications for the catalytic mechanism.the shikimate biosynthetic pathway is essential to microorganisms, plants, and parasites but absent from mammals. therefore, shikimate dehydrogenase (sd) and other enzymes in the pathway are attractive targets for developing nontoxic antimicrobial agents, herbicides, and antiparasite drugs. sd catalyzes the fourth reaction in the pathway, the nicotinamide adenine dinucleotide phosphate- (nadp-) dependent reduction of 3-dehydroshikimic acid to shikimic acid (sa), as well as its reverse, by the tr ...200717649975
identification of a chloride ion binding site in na+/cl -dependent transporters.the recent determination of the crystal structure of the leucine transporter from aquifex aeolicus (aaleut) has provided significant insights into the function of neurotransmitter:sodium symporters. transport by aaleut is cl(-) independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on cl(-) ions. however, the only cl(-) ion identified in the aaleut structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site ...200717652169
thermodynamic stability and folding of proteins from hyperthermophilic organisms.life grows almost everywhere on earth, including in extreme environments and under harsh conditions. organisms adapted to high temperatures are called thermophiles (growth temperature 45-75 degrees c) and hyperthermophiles (growth temperature >or= 80 degrees c). proteins from such organisms usually show extreme thermal stability, despite having folded structures very similar to their mesostable counterparts. here, we summarize the current data on thermodynamic and kinetic folding/unfolding behav ...200717683332
a new sulfurtransferase from the hyperthermophilic bacterium aquifex aeolicus. being single is not so simple when temperature gets high.sulfur is a functionally important element of living matter. rhodanese is involved in the enzymatic production of the sulfane sulfur which has been suggested as the biological relevant active sulfur species. rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla. we characterized a new single-domain rhodanese with a thiosulfate : cyanide transferase activity, aq-477. aq-477 can also use tetrathionate and polysulfide. thermoactivity and thermostability ...200717697123
analysis of glycan polymers produced by peptidoglycan glycosyltransferases.bacterial cells are surrounded by a cross-linked polymer called peptidoglycan, the integrity of which is necessary for cell survival. the carbohydrate chains that form the backbone of peptidoglycan are made by peptidoglycan glycosyltransferases (pgts), highly conserved membrane-bound enzymes that are thought to be excellent targets for the development of new antibacterials. although structural information on these enzymes recently became available, their mechanism is not well understood because ...200717704540
characterization and crystallization of an iscu-type scaffold protein with bound [2fe-2s] cluster from the hyperthermophile, aquifex aeolicus.iscu plays a key role during iron-sulphur (fe-s) cluster biosynthesis as a scaffold for the assembly of a nascent, highly labile fe-s cluster. here we report the characterization of an iscu-type protein (aa iscu) from the hyperthermophilic bacterium aquifex aeolicus. unlike other known homologues of iscu, expression of aa iscu in escherichia coli has yielded an fe-s cluster-containing holo-protein. biochemical and spectroscopic studies of the wild-type aa iscu and its asp38-to-ala substituted (d ...200717846064
split leucine-specific domain of leucyl-trna synthetase from the hyperthermophilic bacterium aquifex aeolicus.leucyl-trna synthetase (leurs) from aquifex aeolicus is the only known heterodimer synthetase. it is named leurs alphabeta;, and its alpha and beta subunits contain 634 and 289 residues, respectively. like thermus thermophilus leurs, leurs alphabeta has a large extra domain, the leucine-specific domain, inserted into the catalytic domain. the subunit split site is exactly in the middle of the leucine-specific domain and may have a unique function. here, a series of mutants of leurs alphabeta con ...200617144674
the core domain of aquifex aeolicus trna (m7g46) methyltransferase has the methyl-transfer activity to trna.transfer rna (m(7)g46) methyltransferase [trmb] catalyses the transfer of methyl groups from s-adenosyl-l-methionine to the n(7)-atom of guanine at position 46 in trna. trmb proteins from thermophilic bacteria such as aquifex aeolicus have a long c-terminal region as compared to those from mesophilic bacteria. further, n-terminal region observed in trmb proteins from mesophiles is missing in a. aeolicus trmb. therefore, we considered that this distinct c-terminal region in a. aeolicus trmb might ...200617150909
binding of uridine 5'-diphosphate in the "basic patch" of the zinc deacetylase lpxc and implications for substrate binding.lpxc is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of lipid a, a vital component of the outer membrane of gram-negative bacteria. accordingly, the inhibition of lpxc is an attractive strategy for the treatment of gram-negative bacterial infections. here, we report the 2.7 a resolution x-ray crystal structure of lpxc from aquifex aeolicus complexed with uridine 5'-diphosphate (udp), and the 3.1 a resolution structure of lpxc complexed with pyrophosphate. the ...200617176043
the substrate specificity of trna (m1g37) methyltransferase (trmd) from aquifex aeolicus.transfer rna (m(1)g37) methyltransferase (trmd) catalyzes methyl-transfer from s-adenosyl-l-methionine to the n(1) atom of g37 in trna. in escherichia coli cells, trmd methylates trna species possessing a g36g37 sequence. it was previously believed that g36 was the positive determinant of trmd recognition. in the current study, we demonstrate that trmd from aquifex aeolicus methylates trna transcripts possessing an a36g37 sequence as well as trna transcripts possessing a g36g37 sequence. in cont ...200617121543
the nucleotide-binding site of aquifex aeolicus lpxc.the structure of recombinant aquifex aeolicus udp-3-o-acyl-n-acetylglucosamine deacetylase (lpxc) in complex with udp has been determined to a resolution of 2.2 a. previous studies have characterized the binding sites of the fatty-acid and sugar moieties of the substrate, udp-(3-o-hydroxymyristoyl)-n-acetylglucosamine, but not that of the nucleotide. the uracil-binding site is constructed from amino acids that are highly conserved across species. hydrophobic associations with the phe155 and arg2 ...200617077484
folding and assembly pathways of co-chaperonin proteins 10: origin of bacterial thermostability.to compare folding/assembly processes of heptameric co-chaperonin proteins 10 (cpn10) from different species and search for the origin of thermostability in hyper-thermostable aquifex aeolicus cpn10 (aacpn10), we have studied two bacterial variants-aacpn10 and escherichia coli cpn10 (groes)-and compared the results to data on homo sapiens cpn10 (hmcpn10). equilibrium denaturation of groes by urea, guanidine hydrochloride (guhcl) and temperature results in coupled heptamer-to-monomer transitions ...200617084377
expression, purification, and characterization of a new heterotetramer structure of leucyl-trna synthetase from aquifex aeolicus in escherichia coli.aminoacyl-trna synthetases are key players in the interpretation of the genetic code. they constitute a textbook example of multi-domain proteins including insertion and terminal functional modules appended to one of the two class-specific active site domains. the non-catalytic domains usually have distinct roles in the aminoacylation reaction. aquifex aeolicus leucyl-trna synthetase (leurs) is composed of a separated catalytic site and trna anticodon-binding site, which would represent one of t ...200616256368
the crystal structure of aq_328 from the hyperthermophilic bacteria aquifex aeolicus shows an ancestral histone fold.the structure of aq_328, an uncharacterized protein from hyperthermophilic bacteria aquifex aeolicus, has been determined to 1.9 a by using multi-wavelength anomalous diffraction (mad) phasing. although the amino acid sequence analysis shows that aq_328 has no significant similarity to proteins with a known structure and function, the structure comparison by using the dali server reveals that it: (1) assumes a histone-like fold, and (2) is similar to an ancestral nuclear histone protein (pdb cod ...200616287087
genetic transplantation: salmonella enterica serovar typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems.in salmonella enterica serovar typhimurium, sigma(28) and anti-sigma factor flgm are regulatory proteins crucial for flagellar biogenesis and motility. in this study, we used s. enterica serovar typhimurium as an in vivo heterologous system to study sigma(28) and anti-sigma(28) interactions in organisms where genetic manipulation poses a significant challenge due to special growth requirements. the chromosomal copy of the s. enterica serovar typhimurium sigma(28) structural gene flia was exchang ...200616352826
insights on a new pdi-like family: structural and functional analysis of a protein disulfide oxidoreductase from the bacterium aquifex aeolicus.a potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been attributed to a new family of protein disulfide isomerase (pdi)-like proteins. members of this family are characterized by a molecular mass of about 26kda and by two trx folds, each comprising a cxxc active site motif. we report on the functional and structural characterization of a new member of this family, which was isolated from the thermophilic bacterium aquifex aeolicus (a ...200616364362
molecular signatures in protein sequences that are characteristics of the phylum aquificae.species of the phylum aquificae are of great interest due to their strict extreme thermophilic growth characteristics. presently, there is no known molecular characteristic which is unique to this group of bacteria. this work describes six conserved inserts and deletions (indels or signature sequences) in four widely distributed proteins that are distinctive features of species from the phylum aquificae. these include three signatures consisting of a 2 aa insert, a 5-6 aa insert and a 6 aa delet ...200616403873
a theoretical model of aquifex pyrophilus flagellin: implications for its thermostability.aquifex pyrophilus is a flagellated hyperthermophilic eubacterial species that grows optimally at 85 degrees c. the thermostable a. pyrophilus flagellar filament is primarily composed of a single protein called flagellin (flaa). the n- and c-terminal sequence regions of flaa are important for self-assembly and share high sequence similarity with mesophilic bacterial flagellins. we have developed a predictive 3d-structure of flaa, using the published structure of mesophilic salmonella typhimurium ...200616411078
dichotomy of major bacterial phyla inferred from gene arrangement comparisons.a computer search for gene arrangements that are present in gram-positive bacteria but are absent from proteobacteria and vice versa was carried out. four such arrangements were detected, based on which major bacterial phyla were divided into two groups; thermotoga, deinococcus-thermus, chloroflexi (green non-sulfur bacteria), and fusobacteria, represent gram-positive bacterial gene arrangements, while aquifex, spirochetes, planctomycetes, chlorobi (green sulfur bacteria), bacteroides show prote ...200616159660
crystal structure of prephenate dehydrogenase from aquifex aeolicus. insights into the catalytic mechanism.the enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. the bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. we report the first crystal structure of a monofunctional prephenate dehydrogen ...200616513644
the slc6 orphans are forming a family of amino acid transporters.transporters in the human genome are grouped in solute carrier families (slc). the slc6 family is one of the biggest transporter families in the human genome comprising 20 members. it is usually referred to as the neurotransmitter transporter family because its founding members encode transporters for the neurotransmitters gaba, noradrenaline, serotonin and dopamine. the family also includes a number of 'orphan' transporters, the function of which has remained elusive until recently. identificat ...200616540203
getting the message across: a recent transporter structure shows the way.efforts to define the mechanisms governing neurotransmitter uptake and drug action have moved into high gear with the publication of a high-resolution structure of a leucine transporter from aquifex aeolicus, a bacterial member of the slc6 transporter family. solved with the substrate leucine bound, the new structure corroborates extensive biochemical and mutagenesis studies performed with related mammalian neurotransmitter transporters and provides exciting suggestions as to how coupling arises ...200616543127
a simple tagging system for protein encapsulation.molecular containers that encapsulate specific cargo can be useful for many natural and non-natural processes. we report a simple system, based on charge complementarity, for the encapsulation of appropriately tagged proteins within an engineered, proteinaceous capsid. four negative charges per monomer were added to the lumazine synthase from aquifex aeolicus (aals). the capsids formed by the engineered aals associate with green fluorescent protein bearing a positively charged deca-arginine tag ...200616594656
nmr structure of the aquifex aeolicus tmrna pseudoknot pk1: new insights into the recoding event of the ribosomal trans-translation.the transfer-messenger rna (tmrna) pseudoknot pk1 is essential for bacterial trans-translation, a ribosomal rescue mechanism. we report the solution structure of pk1 from aquifex aeolicus, which despite an unprecedented small number of nucleotides and thus an unprecented compact size, displays a very high thermal stability. several unusual structural features account for these properties and indicate that pk1 belongs to the class of ribosomal frameshift pseudoknots. this suggests a similarity be ...200616595798
structural and functional studies of the response regulator hupr.hupr is a response regulator that controls the synthesis of the membrane-bound [nife]hydrogenase of the photosynthetic bacterium rhodobacter capsulatus. the protein belongs to the ntrc subfamily of response regulators and is the second protein of a two-component system. we have crystallized the full-length protein hupr in the unphosphorylated state in two dimensions using the lipid monolayer technique. the 3d structure of negatively stained hupr was calculated to a resolution of approximately 23 ...200616631791
flgm anti-sigma factors: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships.flgm proteins, also known as anti-sigma-28 factor (sigma28), are negative regulators of flagellin synthesis. recently, a three-dimensional structure of the aquifex aeolicus sigma28/flgm complex (pdb code: 1rp3) was determined by x-ray crystallography at 2.3 a resolution. furthermore, experimental data on bacterial flgm, including site-directed mutagenesis and structural characterization by nmr are also available. however, an interpretation of the sequence-structure-function relationships combini ...200616673084
the structure of the exopolyphosphatase (ppx) from escherichia coli o157:h7 suggests a binding mode for long polyphosphate chains.polyphosphate (polyp) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. these polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. polyphosphate is synthesized by polyp kinase (ppk), using the terminal phosphate of atp as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. here we report ...200616678853
intersubunit disulfide interactions play a critical role in maintaining the thermostability of glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium aquifex aeolicus.proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. a thermostable glucose-6-phosphate dehydrogenase (tg6pdh) from the hyperthermophilic bacterium aquifex aeolicus was expressed as a recombinant protein in escherichia coli. the a. aeolicus g6pdh is a homodimer exhibiting remarkable thermostability (t1/2 = 24 hr at 90 degrees c). based on homology modeling and upon comparison of its structure with human g6pd ...200616721657
biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium aquifex aeolicus.a monofunctional prephenate dehydrogenase (pd) from aquifex aeolicus was expressed as a his-tagged protein in escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable pd. a. aeolicus pd is susceptible to proteolysis. in this report, the properties of the full-length pd are compared with one of these products, an n-terminally truncated protein variant (delta19pd) also expressed recombinantly in e. coli. b ...200616731976
formation of functional tat translocases from heterologous components.the tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. in eschericha coli, tat transport requires the integral membrane proteins tata, tatb and tatc. in this study we have tested the ability of tat genes from the eubacterial species pseudomonas syringae, streptomyces coelicolor and aquifex aeolicus, to compensate for the absence of the cognate e. coli tat gene, and thus to form functional tat translocases with e. coli tat comp ...200616854235
a comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/na+ symporters (nss) aids in the use of the leut structure to probe nss structure and function.the recently elucidated crystal structure of a prokaryotic member of the neurotransmitter/sodium symporter (nss) family (yamashita et al., 2005) is a major advance toward understanding structure-function relationships in this important class of transporters. to aid in the generalization of these results, we present here a comprehensive sequence alignment of all known prokaryotic and eukaryotic nss proteins, based on the crystal structure of the leucine transporter from aquifex aeolicus (leut). r ...200616880288
a homology model of sert based on the leut(aa) template.a human serotonin transporter (sert) model has been constructed based on the crystal structure of the bacterial homologue of na(+)/cl(-)-dependent neurotransmitter transporters from aquifex aeolicus (leut(aa)). amino acids in the ligand binding area predicted by icm pocket finder included tyr95, ala96, asp98, gly100 (transmembrane helix (tmh) 1), ala169, ile172, ala173, tyr176 (tmh3), phe335, ser336, gly338, phe341, val343 (tmh6), thr439, ala441, and gly442 (tmh8). the present model is an update ...200616919451
multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly.lumazine synthases have been observed in the form of pentamers, dimers of pentamers, icosahedral capsids consisting of 60 subunits and larger capsids with unknown molecular structure. here we describe the analysis of the assembly of native and mutant forms of lumazine synthases from bacillus subtilis and aquifex aeolicus at various ph values and in the presence of different buffers using small angle x-ray scattering and electron microscopy. both wild-type lumazine synthases are able to form caps ...200616935304
comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics.membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. we are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. here, we present the compa ...200616943447
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