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structures of leut in bicelles define conformation and substrate binding in a membrane-like context.neurotransmitter sodium symporters (nsss) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. consistent with the role of nsss in the central nervous system, they are implicated in multiple diseases and disorders. leut, from aquifex aeolicus, is a prokaryotic ortholog of the nss family and has contributed to our understanding of the structure, mechanism and pharmacology of nsss. at present, however, the functional state of leut in crystals gro ...201222245965
spectroscopic characterization of the key catalytic intermediate ni-c in the o2-tolerant [nife] hydrogenase i from aquifex aeolicus: evidence of a weakly bound hydride.ni-c in the o(2)-tolerant hydrogenase i from aquifex aeolicus binds a hydride weaker than that in o(2)-sensitive hydrogenases. this is in line with the enhanced light-sensitivity of ni-c, greater lability of the hydride complex and increased catalytic redox potentials relevant to bio-h(2) oxidation.201122143669
structural insights into the catalytic mechanism of escherichia coli selenophosphate synthetase.selenophosphate synthetase (sps) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-trna. selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. sps activity is dependent on both mg(2+) and k(+) and uses atp, selenide, and water to catalyze the formation of amp, orthophosphate, and selenophosphat ...201222081394
the structure of aquifex aeolicus ribosomal protein s8 reveals a unique subdomain that contributes to an extremely tight association with 16s rrna.the assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. the ribosomal protein s8 from aquifex aeolicus (as8) is unique in that there is a 41-residue insertion in the consensus s8 sequence. in addition, as8 exhibits an unusually high affinity for the 16s ribosomal rna, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than ...201122079365
the cphaii protein from aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity.the cphaii protein from the hyperthermophile aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (mbl) superfamily and presents 28% identity with the aeromonas hydrophila subclass b2 cpha mbl. the gene encoding cphaii was amplified by pcr from the a. aeolicus genomic dna and overexpressed in escherichia coli using a plex-based expression system. the recombinant cphaii protein was purified by a combination of heating (to denature e. coli proteins) and two steps of immobili ...201222009263
dna stretching by bacterial initiators promotes replication origin opening.many replication initiators form higher-order oligomers that process host replication origins to promote replisome formation. in addition to dedicated duplex-dna-binding domains, cellular initiators possess aaa+ (atpases associated with various cellular activities) elements that drive functions ranging from protein assembly to origin recognition. in bacteria, the aaa+ domain of the initiator dnaa has been proposed to assist in single-stranded dna formation during origin melting. here we show cry ...201121964332
NMR structure of an acyl-carrier protein from Borrelia burgdorferi.Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure-determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of ...201121904063
Templates and models of monoamine transporter proteins.X-ray crystallography, structural bioinformatics and computational chemistry have become important techniques in the discovery and development of effective and safe new drugs. From a drug discovery point of view, membrane proteins are among the most interesting molecular targets, but the current knowledge about detailed 3D structures of membrane proteins is sparse. Homology modeling techniques may provide structural knowledge about membrane proteins and their interactions with drugs and other mo ...201121864275
substrate trna recognition mechanism of a multi-site-specific trna methyltransferase, aquifex aeolicus trm1, based on the x-ray crystal structure.archaeal and eukaryotic trna (n(2)-, n(2)-guanine) dimethyltransferase (trm1) produces n(2)-, n(2)-dimethylguanine at position 26 in trna. in contrast, trm1 from aquifex aeolicus, a hyper-thermophilic eubacterium, modifies g27 as well as g26. here, a gel-mobility shift assay revealed that the t-arm in trna is the binding site of a. aeolicus trm1. to address the multi-site specificity, we performed an x-ray crystal structure study. the overall structure of a. aeolicus trm1 is similar to that of a ...201121844194
evidence for a two-metal-ion mechanism in the cytidyltransferase kdsb, an enzyme involved in lipopolysaccharide biosynthesis.lipopolysaccharide (lps) is located on the surface of gram-negative bacteria and is responsible for maintaining outer membrane stability, which is a prerequisite for cell survival. furthermore, it represents an important barrier against hostile environmental factors such as antimicrobial peptides and the complement cascade during gram-negative infections. the sugar 3-deoxy-d-manno-oct-2-ulosonic acid (kdo) is an integral part of lps and plays a key role in lps functionality. prior to its incorpo ...201121826242
peripheral insertion modulates editing activity of the isolated cp1 domain of leucyl-trna synthetase.a large insertion domain called connective peptide 1 (cp1) present in class ia aminoacyl-trna synthetases is responsible for post-transfer editing. leucyl-trna synthetases (leurs) from aquifex aeolicus and giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the cp1 that are crucial for editing activity. crystal structures of aaleurs-cp1 (2.4+à), glleurs-cp1 (2.6+à) and the insertion deletion mutant aaleurs-cp1-+ö20 (2.5+à) were solved to understand the role of thes ...201121819379
crystal structure of prephenate dehydrogenase from streptococcus mutans.prephenate dehydrogenase (pdh) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. this enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. the crystal structure of pdh from streptococcus mutans in a complex with nad(+) shows that the enzyme exists as a homo-dimer, each monomer consisting of tw ...201121798280
crystallization and preliminary x-ray analysis of a putative sensor histidine kinase domain: the c-terminal domain of hksp4 from aquifex aeolicus vf5.the histidine kinase domain of the cytoplasmic protein hksp4 from the hyperthermophilic bacterium aquifex aeolicus vf5, located in the c-terminal half of the protein, was expressed, purified and crystallized. diffraction-quality crystals were obtained in the presence of adenosine triphosphate (atp) or adenosine 5'-(ß,?-imido)triphosphate (amppnp) by the sitting-drop vapour-diffusion method using peg 3350 as the precipitant. the crystals obtained in the presence of atp and amppnp diffracted x-ray ...201121795799
interconversion of functional motions between mesophilic and thermophilic adenylate kinases.dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (ak), for which the open/closed transition limits the rate of catalytic turnover. here, we compare our previously published coarse-grained (double-well go) simulation of mesophilic ak from e. coli (akmeso) to simulations of thermophilic ak from aquifex aeolicus (akthermo). in akthermo, as with akmeso, the lid domain prefers to close before the nmp domain in the presence of ligand, but lid rigid- ...201121779157
The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus.The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3)-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper ...201121738733
survival of thermophilic and hyperthermophilic microorganisms after exposure to uv-c, ionizing radiation and desiccation.in this study, we investigated the ability of several (hyper-) thermophilic archaea and phylogenetically deep-branching thermophilic bacteria to survive high fluences of monochromatic uv-c (254 nm) and high doses of ionizing radiation, respectively. nine out of fourteen tested microorganisms showed a surprisingly high tolerance against ionizing radiation, and two species (aquifex pyrophilus and ignicoccus hospitalis) were even able to survive 20 kgy. therefore, these species had a comparable sur ...201121638055
molecular basis of the differential interaction with lithium of glycine transporters glyt1 and glyt2.j. neurochem. (2011) 10.1111/j.1471-4159.2011.07309.x abstract: glycine synaptic levels are controlled by glycine transporters (glyts) catalyzing na(+) /cl(-) /glycine cotransport. glyt1 displays a 2 : 1 : 1 stoichiometry and is the main regulator of extracellular glycine concentrations. the neuronal glyt2, with higher sodium coupling (3 : 1 : 1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. in this work, using structural homology modelling and molecular dynamics si ...201121574997
structural insight into the rotational switching mechanism of the bacterial flagellar motor.the bacterial flagellar motor can rotate either clockwise (cw) or counterclockwise (ccw). three flagellar proteins, flig, flim, and flin, are required for rapid switching between the cw and ccw directions. switching is achieved by a conformational change in flig induced by the binding of a chemotaxis signaling protein, phospho-chey, to flim and flin. flig consists of three domains, flig(n), flig(m), and flig(c), and forms a ring on the cytoplasmic face of the ms ring of the flagellar basal body. ...201121572987
characterization of a unique [fes] cluster in the electron transfer chain of the oxygen tolerant [nife] hydrogenase from aquifex aeolicus.iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. in the oxygen-tolerant hydrogenase i from aquifex aeolicus such electron "wires" form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of o(2) to water. amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. electron ...201121444783
domain interactions of the transcription-translation coupling factor escherichia coli nusg are intermolecular and transient.the bacterial transcription factor nusg (n-utilization substance g) is suggested to act as a key coupling factor between transcription and translation [burmann, schweimer, luo, wahl, stitt, gottesman and rösch (2010) science 328, 501-504] and contributes to phage ?-mediated antitermination in escherichia coli that enables read-through of early transcription termination sites. e. coli nusg consists of two structurally and functionally distinct domains that are connected through a flexible linker. ...201121345171
conferment of folding ability to a naturally unfolded apocytochrome c through introduction of hydrophobic amino acid residues.hyperthermophilic aquifex aeolicus cytochrome c(555) (aa c(555)) exceptionally folds even in the apo state, unlike general cytochromes c including mesophilic pseudomonas aeruginosa cytochrome c(551) (pa c(551)), which is structurally homologous to aa c(555) in the holo state. here we hypothesized that the exceptional apo aa c(555) folding can be attributed to nine hydrophobic amino acid residues and proved this using a pa c(551) variant (denoted as pa-nh) carrying the nine hydrophobic residues a ...201121329369
purification, crystallization and preliminary x-ray crystallographic analysis of the c-terminal cytoplasmic domain of flhb from aquifex aeolicus.flhb is a key protein in the regulation of protein export by the bacterial flagellar secretion system. it is composed of two domains: an n-terminal transmembrane domain and a c-terminal cytoplasmic domain (flhbc). here, the crystallization and preliminary crystallographic analysis of flhbc from aquifex aeolicus are reported. purified protein was crystallized using the vapour-diffusion technique. the crystals diffracted to 2.3 å resolution and belonged to space group c2, with unit-cell parameters ...201121301106
crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein moga from two thermophilic organisms.molybdenum-cofactor (moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. two proteins, moga and moea, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. here, three crystal structures of the moco-biosynthesis protein moga from the two thermophilic organisms thermus thermophilus (ttmoga; 1.64 å resolution, space group p2(1)) and aquifex aeolicus (aamoga; 1.70 å resolut ...201021206014
control of lipopolysaccharide biosynthesis by ftsh-mediated proteolysis of lpxc is conserved in enterobacteria but not in all gram-negative bacteria.despite the essential function of lipopolysaccharides (lps) in gram-negative bacteria, it is largely unknown how the exact amount of this molecule in the outer membrane is controlled. the first committed step in lps biosynthesis is catalyzed by the lpxc enzyme. in escherichia coli, the cellular concentration of lpxc is adjusted by the only essential protease in this organism, the membrane-anchored metalloprotease ftsh. turnover of e. coli lpxc requires a length- and sequence-specific c-terminal ...201021193611
original design of an oxygen-tolerant [nife] hydrogenase: major effect of a valine-to-cysteine mutation near the active site.hydrogenases are efficient biological catalysts of h(2) oxidation and production. most of them are inhibited by o(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. we have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [nife] hydrogenase from desulfovibrio fructosovorans could impact the reaction of the enzyme with o(2) (dementin, s.; j. am. chem. soc. 2009, 131, 10156-10 ...201021175174
structure of leishmania major methionyl-trna synthetase in complex with intermediate products methionyladenylate and pyrophosphate.leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions of people at risk. due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from leishmania major in hopes of creating a platform for the rational design of new therapeutics. crystals of the catalytic core of methionyl-trna synthetase from l. major (lmmetrs) were obtained with the substrates mgatp and methionine presen ...201021144880
characterization of aquifex aeolicus ribonuclease iii and the reactivity epitopes of its pre-ribosomal rna substrates.ribonuclease iii cleaves double-stranded (ds) structures in bacterial rnas and participates in diverse rna maturation and decay pathways. essential insight on the rnase iii mechanism of dsrna cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, aquifex aeolicus. however, the biochemical properties of a. aeolicus (aa)-rnase iii and the reactivity epitopes of its substrates are not known. the catalytic activity of purified recombinant aa-rnase ...201021138964
discovery of a minimal form of rnase p in pyrobaculum.rnase p rna is an ancient, nearly universal feature of life. as part of the ribonucleoprotein rnase p complex, the rna component catalyzes essential removal of 5' leaders in pre-trnas. in 2004, li and altman computationally identified the rnase p rna gene in all but three sequenced microbes: nanoarchaeum equitans, pyrobaculum aerophilum, and aquifex aeolicus (all hyperthermophiles) [li y, altman s (2004) rna 10:1533-1540]. a recent study concluded that n. equitans does not have or require rnase ...201021135215
assembling networks of microbial genomes using linear programming.microbial genomes exhibit complex sets of genetic affinities due to lateral genetic transfer. assessing the relative contributions of parent-to-offspring inheritance and gene sharing is a vital step in understanding the evolutionary origins and modern-day function of an organism, but recovering and showing these relationships is a challenging problem.201021092133
a naturally chimeric type iia topoisomerase in aquifex aeolicus highlights an evolutionary path for the emergence of functional paralogs.bacteria frequently possess two type iia dna topoisomerases, gyrase and topo iv, which maintain chromosome topology by variously supercoiling, relaxing, and disentangling dna. dna recognition and functional output is thought to be controlled by the c-terminal domain (ctd) of the topoisomerase dna binding subunit (gyra/parc). the deeply rooted organism aquifex aeolicus encodes one type iia topoisomerase conflictingly categorized as either dna gyrase or topo iv. to resolve this enzyme's catalytic ...201021076033
engagement of arginine finger to atp triggers large conformational changes in ntrc1 aaa+ atpase for remodeling bacterial rna polymerase.the ntrc-like aaa+ atpases control virulence and other important bacterial activities through delivering mechanical work to σ54-rna polymerase to activate transcription from σ54-dependent genes. we report the first crystal structure for such an atpase, ntrc1 of aquifex aeolicus, in which the catalytic arginine engages the γ-phosphate of atp. comparing the new structure with those previously known for apo and adp-bound states supports a rigid-body displacement model that is consistent with large- ...201021070941
stabilization role of a phenothiazine derivative on the electrocatalytic oxidation of hydrogen via aquifex aeolicus hydrogenase at graphite membrane electrodes.the [nife] membrane-bound hydrogenase from the microaerophilic, hyperthermophilic aquifex aeolicus bacterium (aa hase) presents oxygen, carbon monoxide, and temperature resistances. since it oxidizes hydrogen with high turnover, this enzyme is thus of particular interest for biotechnological applications, such as biofuel cells. efficient immobilization of the enzyme onto electrodes is however a mandatory step. to gain further insight into the parameters governing the interfacial electron process ...201021043442
new functional sulfide oxidase-oxygen reductase supercomplex in the membrane of the hyperthermophilic bacterium aquifex aeolicus.aquifex aeolicus, a hyperthermophilic and microaerophilic bacterium, obtains energy for growth from inorganic compounds alone. it was previously proposed that one of the respiratory pathways in this organism consists of the electron transfer from hydrogen sulfide (h(2)s) to molecular oxygen. h(2)s is oxidized by the sulfide quinone reductase, a membrane-bound flavoenzyme, which reduces the quinone pool. we have purified and characterized a novel membrane-bound multienzyme supercomplex that bring ...201020971847
structural origins of nitroxide side chain dynamics on membrane protein α-helical sites.understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. epr spectroscopy in combination with site-directed spin labeling (sdsl) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. alt ...201020964375
characterization of c- and n-terminal domains of aquifex aeolicus mutl endonuclease: n-terminal domain stimulates the endonuclease activity of c-terminal domain in a zinc-dependent manner.dna mmr (mismatch repair) is an excision repair system that removes mismatched bases generated primarily by failure of the 3'-5' proofreading activity associated with replicative dna polymerases. mutl proteins homologous to human pms2 are the endonucleases that introduce the entry point of the excision reaction. deficiency in pms2 function is one of the major etiologies of hereditary non-polyposis colorectal cancers in humans. although recent studies revealed that the ctd (c-terminal domain) of ...201120961292
methionyl-trna synthetase from caenorhabditis elegans: a specific multidomain organization for convergent functional evolution.methionyl-trna synthetase (metrs) is a multidomain protein that specifically binds trnamet and catalyzes the synthesis of methionyl-trnamet. the minimal, core enzyme found in aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon-binding domain, which promotes trna-protein association. in eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the trna-protein complexes. eventually, as ...201020954242
the high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter.the structure of the bacterial leucine transporter from aquifex aeolicus (leut(aa)) has been used as a model for mammalian na(+)/cl(-)-dependent transporters, in particular the serotonin transporter (sert). the crystal structure of leut(aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. this is incompatible with the mutually competitive inhibition of substrates and inhibitors of sert. we explored the binding modes of tricyclic antidepressants by h ...201020829432
the oxygen-tolerant hydrogenase i from aquifex aeolicus weakly interacts with carbon monoxide: an electrochemical and time-resolved ftir study.the [nife] hydrogenase (hase i) involved in the aerobic respiration of the hyperthermophilic bacterium aquifex aeolicus shows increased oxygen tolerance and thermostability and can form very stable films on pyrolytic graphite electrodes. oxygen-tolerant enzymes, like the ones from a. aeolicus and ralstonia eutropha, are reported to be insensitive to co inhibition. this is in contrast to known and well-characterized (oxygen-sensitive) hydrogenases, for which carbon monoxide is a competitive inhib ...201020815411
building arks for trna: structure and function of the arc1p family of non-catalytic trna-binding proteins.following the intricate architecture of the eukaryotic cell, protein synthesis involves formation of many macromolecular assemblies, some of which are composed by trna-aminoacylation enzymes. protein-protein and protein-trna interactions in these complexes can be facilitated by non-catalytic trna-binding proteins. this review focuses on the dissection of the molecular, structural and functional properties of a particular family of such proteins: yeast arc1p and its homologues in prokaryotes and ...201020728442
crystal structures of glycinamide ribonucleotide synthetase, purd, from thermophilic eubacteria.glycinamide ribonucleotide synthetase (gar-syn, purd) catalyses the second reaction of the purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine and atp to glycinamide ribonucleotide (gar), adp and pi. in the present study, crystal structures of gar-syn's from thermus thermophilus, geobacillus kaustophilus and aquifex aeolicus were determined in apo forms. crystal structures in ligand-bound forms were also determined for g. kaustophilus and a. aeolicus proteins. in general, ...201020716513
characterizing a monotopic membrane enzyme. biochemical, enzymatic and crystallization studies on aquifex aeolicus sulfide:quinone oxidoreductase.monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. they are endowed with important physiological functions but until now only few of them have been studied. here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxif ...201020691146
new cultural approaches for microaerophilic hyperthermophiles.this article reports on a new culture system designed for studying the effects of nutritional factors on the growth of hyperthermophilic and chemolithotrophic microorganisms. the system comprises 5-l stainless steel jars, an automatic gas dispenser, propylene microplates, and a robotic platform. the culture system was validated using aquifex aeolicus, a hyperthermophilic, chemolithotrophic, and microaerophilic bacterium, which requires hydrogen, oxygen, co₂, and minerals for growth. we demonstra ...201120676678
conformational flexibility of transmembrane helix vii of the human serotonin transporter impacts ion dependence and transport.the serotonin transporter (sert) regulates the serotonin concentration in the synapse and is a target of several antidepressant and psychostimulant drugs. previous work suggested that the middle transmembrane helices (tmhs) of the biogenic amine transporters (tmhs) play a role in substrate and ion recognition. we focused our present studies on exploring the role of tmh vii in transporter function and ion recognition. residues divergent between human sert and drosophila sert (hsert and dsert, res ...201020637736
evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.the unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. ribonuclease hii from the hyperthermophilic archaeon thermococcus kodakaraensis (tk-rnase hii) is stabilized by its remarkably slow unfolding rate, whereas rnase hi from the thermophilic bacterium thermus thermophilus (tt-rnase hi) unfolds rapidly, comparable with to that of rnase hi from escherichia coli (ec-rnase hi).201020615256
crystal structures of bacillus subtilis lon protease.lon atp-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. eubacterial lon proteases contain an n-terminal domain, an atpase domain, and a protease domain, all in one polypeptide chain. the n-terminal domain is thought to be involved in substrate recognition, the atpase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide frag ...201020600124
post-transfer editing by a eukaryotic leucyl-trna synthetase resistant to the broad-spectrum drug an2690.some aarss (aminoacyl-trna synthetases) develop editing mechanisms to correct mis-charged trna. the cp1 (connective peptide 1) domain of leurs (leucyl-trna synthetase) contains the editing active site, which is the proven target for the broad-spectrum drug an2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole). the esi (eukarya-specific insertion 1) in the cp1 domain of glleurs (giardia lamblia leurs) has been identified. similar substitution with the esi from hsleurs (homo sapiens leurs) imp ...201020557293
observation of a chemically labile, noncovalent enzyme intermediate in the reaction of metal-dependent aquifex pyrophilus kdo8ps by time-resolved mass spectrometry.the direct detection of intermediates in enzymatic reactions can yield important mechanistic insights but may be difficult due to short intermediate lifetimes and chemical instability. using a rapid-mixing device coupled with electrospray ionization time-of-flight mass spectrometry, the noncovalent hemiketal intermediate in the reaction of metal-dependent 3-deoxy-d-manno-octulosonate-8-phosphate (kdo8p) synthase from aquifex pyrophilus was observed in the millisecond time range. using single tur ...201020533322
homology modelling of the gaba transporter and analysis of tiagabine binding.a homology model of the human gaba transporter (gat-1) based on the recently reported crystal structures of the bacterial leucine transporter from aquifex aeolicus (leut) was developed. the stability of the resulting model embedded in a membrane environment was analyzed by extensive molecular dynamics (md) simulations. based on docking studies and subsequent md simulations of three compounds, the endogenous ligand gaba and two potent inhibitors, (r)-nipecotic acid and the anti-epilepsy drug tiag ...201020491137
membrane-bound hydrogenase i from the hyperthermophilic bacterium aquifex aeolicus: enzyme activation, redox intermediates and oxygen tolerance.the membrane-bound hydrogenase (hase i) of the hyperthermophilic bacterium aquifex aeolicus belongs to an intriguing class of redox enzymes that show enhanced thermostability and oxygen tolerance. protein film electrochemistry is employed here to portray the interaction of hase i with molecular oxygen and obtain an overall picture of the catalytic activity. fourier transform infrared (ftir) spectroscopy integrated with in situ electrochemistry is used to identify structural details of the [nife] ...201020441192
inhibition of the fe(4)s(4)-cluster-containing protein isph (lytb): electron paramagnetic resonance, metallacycles, and mechanisms.we report the inhibition of the aquifex aeolicus isph enzyme (lytb, (e)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, ec 1.17.1.2) by a series of diphosphates and bisphosphonates. the most active species was an alkynyl diphosphate having ic(50) = 0.45 microm (k(i) approximately 60 nm), which generated a very large change in the 9 ghz epr spectrum of the reduced protein. on the basis of previous work on organometallic complexes, together with computational docking and quantum chemical calc ...201020426416
structural analysis of the extracellular entrance to the serotonin transporter permeation pathway.neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. these transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. a high resolution crystal structure for the monoamine transporters has yet to be solved. we have developed a homology model for the serotonin transporter (sert) based on the crystal structure of the leucine transporter (leut(aa)) from aquifex aeolicus. the ...201020304925
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
"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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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