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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
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
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
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
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
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
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
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
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
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
structure of selenophosphate synthetase essential for selenium incorporation into proteins and rnas.selenophosphate synthetase (sps) catalyzes the activation of selenide with adenosine 5'-triphosphate (atp) to generate selenophosphate, the essential reactive selenium donor for the formation of selenocysteine (sec) and 2-selenouridine residues in proteins and rnas, respectively. many sps are themselves sec-containing proteins, in which sec replaces cys in the catalytically essential position (sec/cys). we solved the crystal structures of aquifex aeolicus sps and its complex with adenosine 5'-(a ...200918773910
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
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
protein ctc from aquifex aeolicus possesses a full-sized 5s rrna-binding domain.ribosomal 5s rna is the only identified target for proteins of the ctc family. all known proteins of this family, except for ctc from aquifex aeolicus, contain a full-sized 5s rrna-binding domain. in the present study a mistake in the published a. aeolicus genome is corrected. it has been demonstrated that the ctc gene of this organism encodes the protein with a full-length 5s rrna-binding domain. this protein binds specifically to the bacterial 5s rrna. thereby, our data show that ctc a. aeolic ...200919041925
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
trna-independent pretransfer editing by class i leucyl-trna synthetase.aminoacyl-trna synthetases catalyze the formation of aminoacyl-trna in a two-step reaction starting with amino acid activation followed by aminoacyl group transfer to trna. to clear mistakes that occasionally occur, some of these enzymes carry out editing activities, acting on the misactivated amino acid (pretransfer editing) or after the transfer on the trna (post-transfer editing). the post-transfer editing pathway of leucyl-trna synthetase has been extensively studied by structural and bioche ...200919068478
dihydroorotase from the hyperthermophile aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.in prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (cps), aspartate transcarbamoylase (atc), and dihydroorotase (dho), are commonly expressed separately and either function independently (escherichia coli) or associate into multifunctional complexes (aquifex aeolicus). in mammals the enzymes are expressed as a single polypeptide chain (cad) in the order cps-dho-atc and associate into a hexamer. this study presents the three-dimensional structure of ...200919128030
adpase activity of recombinantly expressed thermotolerant atpases may be caused by copurification of adenylate kinase of escherichia coli.except for apyrases, atpases generally target only the gamma-phosphate of a nucleotide. some non-apyrase atpases from thermophilic microorganisms are reported to hydrolyze adp as well as atp, which has been described as a novel property of the atpases from extreme thermophiles. here, we describe an apparent adp hydrolysis by highly purified preparations of the aaa+ atpase ntrc1 from an extremely thermophilic bacterium, aquifex aeolicus. this activity is actually a combination of the activities o ...200919143839
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
dynamics in thermotoga neapolitana adenylate kinase: 15n relaxation and hydrogen-deuterium exchange studies of a hyperthermophilic enzyme highly active at 30 degrees c.backbone conformational dynamics of thermotoga neapolitana adenylate kinase in the free form (tnak) and inhibitor-bound form (tnak*ap5a) were investigated at 30 degrees c using (15)n nmr relaxation measurements and nmr monitored hydrogen-deuterium exchange. with kinetic parameters identical to those of escherichia coli ak (ecak) at 30 degrees c, tnak is a unique hyperthermophilic enzyme. these catalytic properties make tnak an interesting and novel model to study the interplay between protein ri ...200919220019
electronic structure of the metal center in the cd(2+), zn(2+), and cu(2+) substituted forms of kdo8p synthase: implications for catalysis.aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (kdo8ps) is active with a variety of different divalent metal ions bound in the active site. the cd(2+), zn(2+), and cu(2+) substituted enzymes display similar values of k(cat) and similar dependence of k(m)(pep) and k(m)(a5p) on both substrate and product concentrations. however, the flux-control coefficients for some of the catalytically relevant reaction steps are different in the presence of zn(2+) or cu(2+), suggesting that ...200919228070
structure and localisation of drug binding sites on neurotransmitter transporters.the dopamine (dat), serotontin (sert) and noradrenalin (net) transporters are molecular targets for different classes of psychotropic drugs. the crystal structure of aquifex aeolicus leut(aa) was used as a template for molecular modeling of dat, sert and net, and two putative drug binding sites (pocket 1 and 2) in each transporter were identified. cocaine was docked into binding pocket 1 of dat, corresponding to the leucine binding site in leut(aa), which involved transmembrane helices (tmhs) 1, ...200919238460
new insights into the respiratory chains of the chemolithoautotrophic and hyperthermophilic bacterium aquifex aeolicus.aquifex aeolicus, a highly hyperthermophilic bacterium, grows chemolithoautotrophically at 85 degrees c, with hydrogen as electron donor and oxygen as electron acceptor in the presence of a sulfur compound. stimulated by its exceptional physiological properties, we have set out to study the oxygen metabolism of this microorganism. with the use of an unconventional integrative proteomic approach combining separation of membrane proteins by blue-native electrophoresis, detection of enzyme activiti ...200919265402
structural basis for binding of rna and cofactor by a ksga methyltransferase.among methyltransferases, ksga and the reaction it catalyzes are conserved throughout evolution. however, the specifics of substrate recognition by the enzyme remain unknown. here we report structures of aquifex aeolicus ksga, in its ligand-free form, in complex with rna, and in complex with both rna and s-adenosylhomocysteine (sah, reaction product of cofactor s-adenosylmethionine), revealing critical structural information on ksga-rna and ksga-sah interactions. moreover, the structures show ho ...200919278652
the crystal structure of aquifex aeolicus prephenate dehydrogenase reveals the mode of tyrosine inhibition.tyra proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. the three tyra subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. the molecular mechanism responsible for tyra substrate selectivity and regulation is unknown. to further our understanding of tyra-catalyzed re ...200919279014
deep sequencing of salmonella rna associated with heterologous hfq proteins in vivo reveals small rnas as a major target class and identifies rna processing phenotypes.the bacterial sm-like protein, hfq, is a key factor for the stability and function of small non-coding rnas (srnas) in escherichia coli. homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as srna-binding proteins has not entirely been clear. to address this, we expressed in salmonella the hfq proteins of two eubacteria (neisseria meningitides, aquifex aeolicus) and an archaeon (methanocaldococcus jannaschii), and analyzed the asso ...200919333007
structural and functional studies of the biotin protein ligase from aquifex aeolicus reveal a critical role for a conserved residue in target specificity.biotin protein ligase (bpl; ec 6.3.4.15) catalyses the formation of biotinyl-5'-amp from biotin and atp, and the succeeding biotinylation of the biotin carboxyl carrier protein. we describe the crystal structures, at 2.4 a resolution, of the class i bpl from the hyperthermophilic bacteria aquifex aeolicus (aabpl) in its ligand-free form and in complex with biotin and atp. the solvent-exposed beta- and gamma-phosphates of atp are located in the inter-subunit cavity formed by the n- and c-terminal ...200919385043
crystallization and preliminary x-ray diffraction analysis of the trna-modification enzyme gida from aquifex aeolicus.the 5-carboxymethylaminomethyl modification of uridine at the first position of the trna anticodon is crucial for accurate protein synthesis by stabilizing the correct codon-anticodon pairing on the ribosome. two conserved enzymes, gida and mnme, are involved in this modification process. aquifex aeolicus gida was crystallized in two different crystal forms: forms i and ii. these crystals diffracted to 3.2 and 2.3 a resolution, respectively, using synchrotron radiation at the photon factory. the ...200919407389
structure of the rna polymerase core-binding domain of sigma(54) reveals a likely conformational fracture point.transcription initiation by bacterial sigma(54)-rna polymerase requires a conformational change of the holopolymerase-dna complex, driven by an enhancer-binding protein. although structures of the core polymerase and the more common sigma(70) factor have been determined, little is known about the structure of the sigma(54) variant. we report here the structure of an aquifex aeolicus sigma(54) domain (residues 69-198), which binds core rna polymerase. the structure is composed of two distinct sub ...200919426742
characterization of membrane-protein interactions for the leucine transporter from aquifex aeolicus by molecular dynamics calculations.multinanosecond molecular dynamics (md) simulations have been employed to characterize the interaction of an integral membrane protein (imp), the leucine transmitter from aquifex aeolicus (yamashita et al., nature 2005, 437, 215-223), with hydrated lipid bilayer membranes in their physiologically relevant liquid crystalline phases. analysis of the md trajectories for dimyristoyl phosphatidylcholine (dmpc), 1-palmitoyl-2-oleoyl phosphatidylcholine (popc), and 1-palmitoyl-2-oleoyl phosphatidyletha ...200919445452
conserved cysteine residues of gida are essential for biogenesis of 5-carboxymethylaminomethyluridine at trna anticodon.the 5-carboxymethylaminomethyl modification of uridine (cmnm(5)u) at the anticodon first position occurs in trnas that read split codon boxes ending with purine. this modification is crucial for correct translation, by restricting codon-anticodon wobbling. two conserved enzymes, gida and mnme, participate in the cmnm(5)u modification process. here we determined the crystal structure of aquifex aeolicus gida at 2.3 a resolution. the structure revealed the tight interaction of gida with fad. struc ...200919446527
safety evaluation of 1,4-alpha-glucan branching enzymes from bacillus stearothermophilus and aquifex aeolicus expressed in bacillus subtilis.1,4-alpha-glucan branching enzyme (be; ec 2.4.1.18) is a key biocatalyst in the synthesis of polysaccharides, and is therefore useful in the production of food ingredients. the bes evaluated in this study (be-01 and be-02) are obtained by fermentation of bacillus subtilis expressing the be gene from either bacillus stearothermophilus strain trbe14 or aquifex aeolicus strain vf5. the safety of be-01 and be-02 have not been previously evaluated, and therefore, both were subjected to standard toxic ...200919470400
the structure of aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration.sulfide:quinone oxidoreductase (sqr) is a flavoprotein with homologues in all domains of life except plants. it plays a physiological role both in sulfide detoxification and in energy transduction. we isolated the protein from native membranes of the hyperthermophilic bacterium aquifex aeolicus, and we determined its x-ray structure in the "as-purified," substrate-bound, and inhibitor-bound forms at resolutions of 2.3, 2.0, and 2.9 a, respectively. the structure is composed of 2 rossmann domains ...200919487671
aquifex aeolicus trna (n2,n2-guanine)-dimethyltransferase (trm1) catalyzes transfer of methyl groups not only to guanine 26 but also to guanine 27 in trna.transfer rna (n2,n2-guanine)-dimethyltransferase (trm1) catalyzes n2,n2-dimethylguanine formation at position 26 (m(2)(2)g26) in trna. in the reaction, n2-guanine at position 26 (m(2)g26) is generated as an intermediate. the trm1 genes are found only in archaea and eukaryotes, although it has been reported that aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. to confirm whether a. aeolicus trm1 has trna methyltransferase activity, we purified recombinant trm1 protein ...200919491098
generation of a 3d model for human gaba transporter hgat-1 using molecular modeling and investigation of the binding of gaba.a three-dimensional model of the human na(+)/cl(-)-dependent gamma-aminobutyric acid (gaba) transporter hgat-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from aquifex aeolicus (leut(aa)) as the template. protein structure quality checks show that the resulting structure is particularly suited for the analysis of the substrate binding pocket and virtual screening experiments. interactions ...200919506924
insights into trna-dependent amidotransferase evolution and catalysis from the structure of the aquifex aeolicus enzyme.many bacteria form gln-trna(gln) and asn-trna(asn) by conversion of the misacylated glu-trna(gln) and asp-trna(asn) species catalyzed by the gatcab amidotransferase in the presence of atp and an amide donor (glutamine or asparagine). here, we report the crystal structures of gatcab from the hyperthermophilic bacterium aquifex aeolicus, complexed with glutamine, asparagine, aspartate, adp, or atp. in contrast to the staphylococcus aureus gatcab, the a. aeolicus enzyme formed acyl-enzyme intermedi ...200919520089
waaa of the hyperthermophilic bacterium aquifex aeolicus is a monofunctional 3-deoxy-d-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.the hyperthermophile aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. although it has long been recognized that this unique gram-negative bacterium carries genes for different steps of lipopolysaccharide (lps) formation, data on the lps itself or detailed knowledge of the lps pathway beyond the first committed steps of lipid a and 3-deoxy-d-manno-oct-2-ulosonic acid (kdo) synthesis are still lacking. we now report the functional characterization of the thermostable kdo ...200919546212
hyperstability and crystal structure of cytochrome c(555) from hyperthermophilic aquifex aeolicus.in order to elucidate the relationship between the stability and the structure of the monohaem cytochrome c(555) (aa c(555)) from the hyperthermophilic bacterium aquifex aeolicus, chemical denaturation and crystal structure determination were carried out. aa c(555) exhibited higher stability than the thermophilic hydrogenobacter thermophilus cytochrome c(552) (ht c(552)), which is one of the most stable cytochromes c. the three-dimensional crystal structure of aa c(555), which was determined usi ...200919622864
immobilization of the hyperthermophilic hydrogenase from aquifex aeolicus bacterium onto gold and carbon nanotube electrodes for efficient h2 oxidation.the electrochemistry of membrane-bound [nife] hydrogenase i ([nife]-hase i) from the hyperthermophilic bacterium aquifex aeolicus was investigated at gold and graphite electrodes. direct and mediated h(2) oxidation were proved to be efficient in a temperature range of 25-70 degrees c, describing a potential window for h(2) oxidation similar to that of o(2)-tolerant hydrogenases. search for enhancement of current densities and enzyme stability was achieved by the use of carbon nanotube coatings. ...200919629542
a unique beta-1,2-mannosyltransferase of thermotoga maritima that uses di-myo-inositol phosphate as the mannosyl acceptor.in addition to di-myo-inositol-1,3'-phosphate (dip), a compatible solute widespread in hyperthermophiles, the organic solute pool of thermotoga maritima comprises 2-(o-beta-d-mannosyl)-di-myo-inositol-1,3'-phosphate (mdip) and 2-(o-beta-d-mannosyl-1,2-o-beta-d-mannosyl)-di-myo-inositol-1,3'-phosphate (mmdip), two newly identified beta-1,2-mannosides. in cells grown under heat stress, mdip was the major solute, accounting for 43% of the total pool; mmdip and dip accumulated to similar levels, eac ...200919648237
receiver domains control the active-state stoichiometry of aquifex aeolicus sigma54 activator ntrc4, as revealed by electrospray ionization mass spectrometry.a common challenge with studies of proteins in vitro is determining which constructs and conditions are most physiologically relevant. sigma(54) activators are proteins that undergo regulated assembly to form an active atpase ring that enables transcription by sigma(54)-polymerase. previous studies of aaa(+) atpase domains from sigma(54) activators have shown that some are heptamers, while others are hexamers. because active oligomers assemble from off-state dimers, it was thought that even-numb ...200919699748
prokaryotic homologs of argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements.in eukaryotes, rna interference (rnai) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mrnas. the rnai systems recognize the target rna molecules via small guide rnas that are completely or partially complementary to a region of the target. key components of the rnai systems are proteins of the argonaute-piwi family some of which function as slicers, the nucleases that cleave the target rna that is base-paired to a guide r ...200919706170
the structure of eukaryotic and prokaryotic complex i.the structures of the nadh dehydrogenases from bos taurus and aquifex aeolicus have been determined by 3d electron microscopy, and have been analyzed in comparison with the previously determined structure of complex i from yarrowia lipolytica. the results show a clearly preserved domain structure in the peripheral arm of complex i, which is similar in the bacterial and eukaryotic complex. the membrane arms of both eukaryotic complexes show a similar shape but also significant differences in dist ...201019732833
heme is not required for aquifex aeolicus cytochrome c(555) polypeptide folding.in cytochrome c, it has been supposed that heme must bind to the apo polypeptide for structure formation. we constructed a c12a/c15a variant of hyperthermophilic aquifex aeolicus cytochrome c(555) (aa c(555)) in which the covalently heme-binding cys residues were replaced by ala, and characterized its molecular features. the apo c12a/c15a variant had almost the same helical content as holo aa c(555), and spontaneously incorporated heme in vitro with no helical content change. these results sugge ...200919734675
mechanism for the definition of elongation and termination by the class ii cca-adding enzyme.the cca-adding enzyme synthesizes the cca sequence at the 3' end of trna without a nucleic acid template. the crystal structures of class ii thermotoga maritima cca-adding enzyme and its complexes with ctp or atp were determined. the structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related aquifex aeolicus a-adding enzyme by the corresponding domains of the t. maritima cca-adding enzyme allowed the a-adding enzyme to ...200919745807
adenosine triphosphate stimulates aquifex aeolicus mutl endonuclease activity.background: human pms2 (hpms2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack muth. mn(++) was previously found to stimulate the endonuclease activity of these homologues. atp was required for the nicking activity of hpms2 and ypms1, but was reported to inhibit bacterial mutl proteins from thermus thermophilus and aquifex aeolicus that displayed homology to hpms2. mutational analysis has identified the dqha(x)(2)e(x)(4)e motif presen ...200919777055
a soluble recn homologue provides means for biochemical and genetic analysis of dna double-strand break repair in escherichia coli.recn is a highly conserved, smc-like protein in bacteria. it plays an important role in the repair of dna double-strand breaks and is therefore a key factor in maintaining genome integrity. the insolubility of escherichia coli recn has limited efforts to unravel its function. we overcame this limitation by replacing the resident coding sequence with that of haemophilus influenzae recn. the heterologous construct expresses haemophilus recn from the sos-inducible e. coli promoter. the hybrid gene ...200919846353
discovery and characterization of three new escherichia coli septal ring proteins that contain a spor domain: damx, dedd, and rlpa.spor domains are approximately 70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. the spor domains in the ftsn cell division proteins from escherichia coli and caulobacter crescentus have been shown to bind peptidoglycan. besides ftsn, e. coli has three additional spor domain proteins--damx, dedd, and rlpa. we show here that all three of these proteins localize to the septal ring in e. coli. the loss of damx or dedd either alone or in combination with ...201019880599
trna-dependent pre-transfer editing by prokaryotic leucyl-trna synthetase.to prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-trna synthetases have evolved editing activities to eliminate intermediate or final non-cognate products. in this work we studied the different editing pathways of class ia leucyl-trna synthetase (leurs). different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound an2690 that targets the post-transfer editing site of leurs. t ...201019940155
emerging structure-function relationships defining monoamine nss transporter substrate and ligand affinity.monoamine transporters are a group of transmembrane neurotransmitter sodium symporter (nss) transporters that play a crucial role in regulating biogenic monoamine concentrations at peripheral and central synapses. given the key role played by serotonin, dopamine and noradrenaline in addictive and disease states, structure-function studies have been conducted to help guide the development of improved central nervous system therapeutics. extensive pharmacological, immunological and biochemical stu ...201019954741
structural biology of s-adenosylmethionine decarboxylase.s-adenosylmethionine decarboxylase (adometdc) is a critical enzyme in the polyamine biosynthetic pathway and a subject of many structural and biochemical investigations for anti-cancer and anti-parasitic therapy. the enzyme undergoes an internal serinolysis reaction as a post-translational modification to generate the active site pyruvoyl group for the decarboxylation process. the crystal structures of adometdc from homo sapiens, solanum tuberosum, thermotoga maritima, and aquifex aeolicus have ...201019997761
structure of sure protein from aquifex aeolicus vf5 at 1.5 a resolution.sure is a stationary-phase survival protein found in bacteria, eukaryotes and archaea that exhibits a divalent-metal-ion-dependent phosphatase activity and acts as a nucleotidase and polyphosphate phosphohydrolase. the structure of the sure protein from the hyperthermophile aquifex aeolicus has been solved at 1.5 a resolution using molecular replacement with one dimer in the asymmetric unit and refined to an r factor of 15.6%. the crystal packing reveals that two dimers assemble to form a tetram ...200920054112
structure of d-lactate dehydrogenase from aquifex aeolicus complexed with nad(+) and lactic acid (or pyruvate).the crystal structure of d-lactate dehydrogenase from aquifex aeolicus (aq_727) was determined to 2.12 a resolution in space group p2(1)2(1)2(1), with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 a. the structure was solved by molecular replacement using the coenzyme-binding domain of lactobacillus helveticus d-lactate dehydrogenase and contained two homodimers in the asymmetric unit. each subunit of the homodimer was found to be in a ;closed' conformation with the nadh cofactor bound t ...200920054113
characterization of a new periplasmic single-domain rhodanese encoded by a sulfur-regulated gene in a hyperthermophilic bacterium aquifex aeolicus.rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. rhodanese domains occur in the three major domains of life. we have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, aquifex aeolicus, with thiosulfate:cyanide transferase activity, aq-1599. the oligomeric organization of the enzyme is stabilized by a disu ...201020060433
a new structure-based classification of sulfide:quinone oxidoreductases.sulfide:quinone oxidoreductases (sqr) are ubiquitous membrane-bound flavoproteins involved in sulfide detoxification, in sulfide-dependent energy conservation processes and potenatially in the homeostasis of the neurotransmitter sulfide. the first 2 structures of sqrs from the bacterium aquifex aeolicus (marcia et al., proc natl acad sci usa 2009; 106:9625-9630) and the archaeon acidianus ambivalens (brito et al., biochemistry 2009; 48:5613-5622) were determined recently by x-ray crystallography ...201020077566
substrate-dependent proton antiport in neurotransmitter:sodium symporters.neurotransmitter-sodium symporters (nss), targets for psychostimulants and therapeutic drugs, have a critical role in neurotransmission. whereas eukaryotic nss show chloride-dependent transport, bacterial nss feature cl(-)-independent substrate transport. recently we showed that mutation of an acidic residue near one of the sodium ion-binding sites in leut of aquifex aeolicus or tyt1 of fusobacterium nucleatum renders substrate binding and/or transport cl(-) dependent. we reasoned that the negat ...201020081826
free and atp-bound structures of ap4a hydrolase from aquifex aeolicus v5.asymmetric diadenosine tetraphosphate (ap(4)a) hydrolases degrade the metabolite ap(4)a back into atp and amp. the three-dimensional crystal structure of ap(4)a hydrolase (16 kda) from aquifex aeolicus has been determined in free and atp-bound forms at 1.8 and 1.95 a resolution, respectively. the overall three-dimensional crystal structure of the enzyme shows an alphabetaalpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. the atp molecul ...201020124691
expression, purification and x-ray analysis of 1,3-propanediol dehydrogenase (aq_1145) from aquifex aeolicus vf5.1,3-propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-propanediol to 3-hydroxypropanal with the simultaneous reduction of nadp(+) to nadph. semet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium aquifex aeolicus vf5 was overexpressed in escherichia coli and purified to homogeneity. crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. single-wavelength data were collected at the selenium pea ...201020124718
a human pms2 homologue from aquifex aeolicus stimulates an atp-dependent dna helicase.mismatch repair in escherichia coli involves a number of proteins including mutl and uvrd. eukaryotes also possess mutl homologues; however, no uvrd helicase homologues have been identified. the hyperthermophilic bacterium aquifex aeolicus has a mutl protein (aae mutl) that possesses a latent endonuclease activity similar to eukaryotic, but different from e. coli, mutl proteins. by sequence homology aq793 is a member of the pcra/uvrd/rep helicase subfamily. we expressed aae mutl and the putative ...201020129926
bioorganometallic mechanism of action, and inhibition, of isph.we have investigated the mechanism of action of aquifex aeolicus isph [e-4-hydroxy-3-methyl-but-2-enyl diphosphate (hmbpp) reductase], together with its inhibition, using a combination of site-directed mutagenesis (k ( m ),v (max)), epr and (1)h, (2)h, (13)c, (31)p, and (57)fe-electron-nuclear double resonance (endor) spectroscopy. on addition of hmbpp to an (unreactive) e126a isph mutant, a reaction intermediate forms that has a very similar epr spectrum to those seen previously with the hmbpp ...201020173096
effects of buffer loading for electrospray ionization mass spectrometry of a noncovalent protein complex that requires high concentrations of essential salts.electrospray ionization (esi) mass spectrometry (ms) is a powerful method for analyzing the active forms of macromolecular complexes of biomolecules. however, these solutions often contain high concentrations of salts and/or detergents that adversely effect esi performance by making ion formation less reproducible, causing severe adduction or ion suppression. many methods for separating complexes from nonvolatile additives are routinely used with esi-ms, but these methods may not be appropriate ...201020226685
"two-step" chronoamperometric method for studying the anaerobic inactivation of an oxygen tolerant nife hydrogenase.hydrogenases catalyze the oxidation and production of h(2). the fact that they could be used in biotechnological devices if they resisted inhibition by o(2) motivates the current research on their inactivation mechanism. direct electrochemistry has been thoroughly used in this respect but often in a qualitative manner. we propose a new and precise chronoamperometric method for studying the anaerobic inactivation mechanism of hydrogenase, which we apply to the oxygen-tolerant nife enzyme from aqu ...201020230028
aquifex aeolicus flgm protein exhibits a temperature-dependent disordered nature.studies on the nature and function of intrinsically disordered proteins (idp) over the past 10 years have demonstrated the importance of idps in normal cellular function. although many proteins predicted to be idps have been experimentally characterized on an individual basis, the conservation of disorder between homologous proteins from different organisms has not been fully studied. we now demonstrate that the flgm protein from the thermophile aquifex aeolicus exhibits a more ordered conformat ...201020298817
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