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thermophilic archaeal enzymes and applications in biocatalysis.thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. they can be used as biocatalysts in the application of 'green chemistry'. the thermophilic archaea contain enzymes that have already been used in commercial applications such as the l-aminoacylase from thermococcus litoralis for the resolution of amino acids and amino acid analogues. this enzyme differs from bacterial l-a ...201121265764
stability of diether c(25,25) liposomes from the hyperthermophilic archaeon aeropyrum pernix k1.temperature and ph effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the aeropyrum pernix. we determined the permeability of c(25,25) liposomes using fluorescence intensity of released calcein. at ph 7.0 and 9.0, and from 85°c to 98°c, only 10% of entrapped calcein was released. after 10 h at 90°c, calcein release reached 27%, independent of ph. fluorescence anisotropy measurements of hydrophobic ...201121295560
cooperativity of alpha- and beta-subunits of group ii chaperonin from the hyperthermophilic archaeum aeropyrum pernix k1.alpha- and beta-subunits (apcpna and apcpnb) are group ii chaperonins from the hyperthermophilic archaeum aeropyrum pernix k1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. in the present study, the cooperativity of alpha- and beta-subunits from the a. pernix k1 was investigated. the apcpna and apcpnb chaperonin genes were overexpressed in e. coli rosetta and codonplus (de3), respectively. each of the recombinant alpha ...201121364306
purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon aeropyrum pernix.the bioenergetics of archaea with respect to the evolution of electron transfer systems is very interesting. in contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from archaea. in particular, c-type cytochromes have been reported only for a limited number of species.201121396131
microbial community structure of hydrothermal deposits from geochemically different vent fields along the mid-atlantic ridge.to evaluate the effects of local fluid geochemistry on microbial communities associated with active hydrothermal vent deposits, we examined the archaeal and bacterial communities of 12 samples collected from two very different vent fields: the basalt-hosted lucky strike (37°17'n, 32°16.3'w, depth 1600-1750 m) and the ultramafic-hosted rainbow (36°13'n, 33°54.1'w, depth 2270-2330 m) vent fields along the mid-atlantic ridge (mar). using multiplexed barcoded pyrosequencing of the variable region 4 ...201121418499
lipid-dependent gating of a voltage-gated potassium channel.recent studies hypothesized that phospholipids stabilize two voltage-sensing arginine residues of certain voltage-gated potassium channels in activated conformations. it remains unclear how lipids directly affect these channels. here, by examining the conformations of the kvap in different lipids, we showed that without voltage change, the voltage-sensor domains switched from the activated to the resting state when their surrounding lipids were changed from phospholipids to nonphospholipids. suc ...201121427721
stability of heterochiral hybrid membrane made of bacterial sn-g3p lipids and archaeal sn-g1p lipids.the structure of membrane lipids in archaea is different from those of bacteria and eucarya in many ways including the chirality of the glycerol backbone. until now, heterochiral membranes were believed to be unstable; thus, no cellular organism could have existed before the separation of the groups of life. in this study, we tested the formation of heterochiral hybrid membrane made of bacterial sn-glycerol-3-phosphate-type polar lipid and archaeal sn-glycerol-1-phosphate-type polar lipid using ...201121473653
discovery of permuted and recently split transfer rnas in archaea.abstract: background: as in eukaryotes, precursor transfer rnas in archaea often contain introns that are removed in trna maturation. two unrelated archaeal species display unique pre-trna processing complexity in the form of split trna genes, in which 2-3 segments of trnas are transcribed from different loci, then trans-spliced to form a mature trna. another rare type of pre-trna, found only in eukaryotic algae, is permuted, where the 3' half is encoded upstream of the 5' half, and must be proc ...201121489296
artificial modulation of the gating behavior of a k channel in a kvap-dna chimera.we present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. we construct a channel-dna chimera with the kvap potassium channel reconstituted in an artificial membrane. the channel is functional and the single channel ion conductivity unperturbed by the presence of the dna. however, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the dna ...201121526187
a synthetic s6 segment derived from kvap channel self-assembles, permeabilizes lipid vesicles and also exhibits ion channel activity in bilayer lipid membrane.kvap is a voltage gated tetrameric potassium channel with six transmembrane (tm) (s1-s6) segments in each monomer from an archaeon, aeropyrum pernix. the objective of the present investigation was to understand the plausible role of the s6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of kvap channel. for this purpose a 22-residue peptide, corresponding to the s6 tm segment of kvap (amino acid 218-239) and a scrambled pe ...201121592970
enhanced activity and enantioselectivity of a hyperthermophilic esterase from archaeon aeropyrum pernix k1 by acetone treatment.to improve the activity and enantioselectivity of hyperthermophilic archaeon aeropyrum pernix k1 esterase (ape1547) and its mutants, they were purified by acetone-treated method. it was found that the acetone treatment not only caused ape1547 and its mutants to display higher activity and enantioselectivity but also saved more than 90% of time spent in purifying them by ni-chelating column. in hydrolysis of p-nitrophenyl caprylate, the acetone-treated ape1547 and mutant a containing the followin ...201121643808
provirus induction in hyperthermophilic archaea: characterization of aeropyrum pernix spindle-shaped virus 1, apsv1, and aeropyrum pernix ovoid virus 1, apov1.by in silico analysis we have identified two putative proviruses in the genome of the hyperthermophilic archaeaon aeropyrum pernix and in special conditions of a. pernix growth we were able to induce their replication. both viruses were isolated and characterized. negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180-210 nm × 40-55 nm, with tails of heterogeneous length, in the range of 0-300 nm. this virus was named aeropyrum pernix spindle-shaped virus 1 ...201121784945
switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering.the inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apaap) from the hyperthermophilic archaeon aeropyrum pernix k1 into a specific carboxylesterase by making a single mutation. in order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apaap can be further changed ...201121748600
Crystallization and preliminary X-ray analysis of a dye-linked D-lactate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix.A dye-linked D-lactate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 63.4, b = 119.4, c = 70.2 Å, ß = 112.0°, and diffracted to 2.0 Å resolution on the BL26B1 beamline at SPring-8. The overall R(merge) was 4.5% and the completeness was 99.8%.201122102248
cleavage of intron from the standard or non-standard position of the precursor trna by the splicing endonuclease of aeropyrum pernix, a hyper-thermophilic crenarchaeon, involves a novel rna recognition site in the crenarchaea specific loop.in crenarchaea, several trna genes are predicted to express precursor-trnas (pre-trnas) with canonical or non-canonical introns at various positions. we initially focused on the trna(thr) species of hyperthermophilic crenarchaeon, aeropyrum pernix (ape) and found that in the living ape cells three trna(thr) species were transcribed and subsequently matured to functional trnas. during maturation, introns in two of them were cleaved from standard and non-standard positions. biochemical studies rev ...201121846775
characterization of family iv udg from aeropyrum pernix and its application in hot-start pcr by family b dna polymerase.recombinant uracil-dna glycosylase (udg) from aeropyrum pernix (a. pernix) was expressed in e. coli. the biochemical characteristics of a. pernix udg (apeudg) were studied using oligonucleotides carrying a deoxyuracil (du) base. the optimal temperature range and ph value for du removal by apeudg were 55-65°c and ph 9.0, respectively. the removal of du was inhibited by the divalent ions of zn, cu, co, ni, and mn, as well as a high concentration of nacl. the opposite base in the complementary stra ...201122087273
structure and characterization of the 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from aeropyrum pernix.the first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dah7ps), varies significantly in size and complexity in the bacteria and plants that express it. the dah7ps from the archaebacterium aeropyrum pernix (dah7ps(ap)) is among the smallest and least complex of the dah7ps enzymes, leading to the hypothesis that dah7ps(ap) would not be subject to feedback regulation by shikimic acid pathway products. we overexpressed dah7ps(ap) in escheric ...201122035970
profile structures of the voltage-sensor domain and the voltage-gated k^{+}-channel vectorially oriented in a single phospholipid bilayer membrane at the solid-vapor and solid-liquid interfaces determined by x-ray interferometry.one subunit of the prokaryotic voltage-gated potassium ion channel from aeropyrum pernix (kvap) is comprised of six transmembrane α helices, of which s1-s4 form the voltage-sensor domain (vsd) and s5 and s6 contribute to the pore domain (pd) of the functional homotetramer. however, the mechanism of electromechanical coupling interconverting the closed-to-open (i.e., nonconducting-to-k^{+}-conducting) states remains undetermined. here, we have vectorially oriented the detergent (og)-solubilized v ...201122060407
In vitro folding of K(v)AP, a voltage-gated K(+) channel.In this contribution, we report in vitro folding of the archaebacterial voltage-gated K(+) channel, K(v)AP. We show that in vitro folding of the K(v)AP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature, and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature, a ...201122044112
crystal structure of peroxiredoxin from aeropyrum pernix k1 complexed with its substrate, hydrogen peroxide.peroxiredoxin (prx) reduces hydrogen peroxide and alkyl peroxides to water and corresponding alcohols, respectively. the reaction is dependent on a peroxidatic cysteine, whose sulphur atom nucleophilically attacks one of the oxygen atoms of the peroxide substrate. in spite of the many structural studies that have been carried out on this reaction, the tertiary structure of the hydrogen peroxide-bound form of prx has not been elucidated. in this paper, we report the crystal structure of prx from ...201019819903
expression and characterization of thymine-dna glycosylase from aeropyrum pernix.the recombinant thymine-dna glycosylase (tdg) from aeropyrum pernix (a. pernix) was expressed in escherichia coli. the enzymatic activity of recombinant a. pernix tdg (apetdg) was characterized using oligonucleotides containing a thymine/uracil base as substrate. apetdg had distinct glycosylase activity on t/g mismatch. the optimal temperature and ph for thymine removal were 65-70 degrees c and ph 7.0-8.5, respectively. high concentration of nacl inhibited the thymine removal. divalent ions had ...201019825417
thermodynamic and kinetic stability of a large multi-domain enzyme from the hyperthermophile aeropyrum pernix.the multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile aeropyrum pernix was studied by denaturant-induced unfolding. at ph 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at ph 3.0, an apparently two-state and highly reversible unfolding occurred. analytical ultracentrifugation revealed the dissociation from dimer to monomer at ph 3.0. the thermodynamic and kinetic stability were studied at ph 3.0 to exp ...201020058042
the terminal 5' phosphate and proximate phosphorothioate promote ligation-independent cloning.function studies of many proteins are waited to develop after genome sequencing. high-throughout technology of gene cloning will strongly promote proteins' function studies. here we describe a ligation-independent cloning (lic) method, which is based on the amplification of target gene and linear vector by pcr using phosphorothioate-modified primers and the digestion of pcr products by lambda exonuclease. the phosphorothioate inhibits the digestion and results in the generation of 3' overhangs, ...201020217896
lipid-protein nanodiscs as reference medium in detergent screening for high-resolution nmr studies of integral membrane proteins.the choice of a suitable detergent-based membrane mimetic is of crucial importance for high-resolution nmr studies of membrane proteins. the present report describes a new approach of detergent screening. it is based on the comparison of 2d (1)h,(15)n-correlation spectra of a protein in a membrane-bilayer "reference" medium and in "trial" detergent-based environments. the proposed "reference" medium is the lipid-protein nanodisc (lpn) representing nanoscale phospholipid bilayers wrapped around b ...201020356311
nmr structural and dynamical investigation of the isolated voltage-sensing domain of the potassium channel kvap: implications for voltage gating.the structure and dynamics of the isolated voltage-sensing domain (vsd) of the archaeal potassium channel kvap was studied by high-resolution nmr. the almost complete backbone resonance assignment and partial side-chain assignment of the (2)h,(13)c,(15)n-labeled vsd were obtained for the protein domain solubilized in dpc/ldao (2:1) mixed micelles. secondary and tertiary structures of the vsd were characterized using secondary chemical shifts and noe contacts. these data indicate that the spatial ...201020356312
overexpression, purification, and characterization of beta-subunit group ii chaperonin from hyperthermophilic aeropyrum pernix k1.in the present study, overexpression, purification, and characterization of aeropyrum pernix k1 chaperonin b in e. coli were investigated. the chaperonin beta-subunit gene (apcpnb, 1,665 bp orf) from the hyperthermophilic archaeon a. pernix k1 was amplified by pcr and subcloned into vector pet21a. the constructed pet21a-apcpnb (6.9 kb) was transformed into e. coli bl21 codonplus (de3). the transformant cell successfully expressed apcpnb, and the expression of apcpnb (61.2 kda) was identified thr ...201020372025
diversity of viruses of the hyperthermophilic archaeal genus aeropyrum, and isolation of the aeropyrum pernix bacilliform virus 1, apbv1, the first representative of the family clavaviridae.we have surveyed the morphological diversity of viruses infecting the archaeon aeropyrum pernix, the most thermophilic species among aerobic organisms, growing optimally at 90 degrees c, and isolated and characterized a novel virus, aeropyrum pernix bacilliform virus 1, apbv1. this is the first virus to be described of the genus aeropyrum and the archaeal order desulfurococcales. the virion of apbv1 has rigid bacilliform morphology, about 140x20nm, with one end pointed and the other rounded. it ...201020430412
an efficient method for the expression and reconstitution of thermostable mn/fe superoxide dismutase from aeropyrum pernix k1.the gene ape0743 encoding the superoxide dismutase (apsod) of a hyperthermophilic archaeon aeropyrum pernix k1 was cloned and over-expressed as a gst fusion protein at a high level in escherichia coli. the expressed protein was simply purified by the process of glutathione affinity chromatography and thrombin treatment. the apsod was a homodimer of 25 kda subunits and a cambialistic sod which was active with either fe(ii) or mn(ii) as a cofactor. the apsod was highly stable against high temperat ...201020467245
down-state model of the voltage-sensing domain of a potassium channel.voltage-sensing domains (vsds) of voltage-gated potassium (kv) channels undergo a series of conformational changes upon membrane depolarization, from a down state when the channel is at rest to an up state, all of which lead to the opening of the channel pore. the crystal structures reported to date reveal the pore in an open state and the vsds in an up state. to gain insights into the structure of the down state, we used a set of experiment-based restraints to generate a model of the down state ...201020550898
solution structure and phospholipid interactions of the isolated voltage-sensor domain from kvap.voltage-sensor domains (vsds) are specialized transmembrane segments that confer voltage sensitivity to many proteins such as ion channels and enzymes. the activities of these domains are highly dependent on both the chemical properties and the physical properties of the surrounding membrane environment. to learn about vsd-lipid interactions, we used nuclear magnetic resonance spectroscopy to determine the structure and phospholipid interface of the vsd from the voltage-dependent k(+) channel kv ...201020851706
structural basis for mrna surveillance by archaeal pelota and gtp-bound ef1α complex.no-go decay and nonstop decay are mrna surveillance pathways that detect translational stalling and degrade the underlying mrna, allowing the correct translation of the genetic code. in eukaryotes, the protein complex of pelota (yeast dom34) and hbs1 translational gtpase recognizes the stalled ribosome containing the defective mrna. recently, we found that archaeal pelota (apelota) associates with archaeal elongation factor 1α (aef1α) to act in the mrna surveillance pathway, which accounts for t ...201020876129
a novel class of protease targets of phosphatidylethanolamine-binding proteins (pebp): a study of the acylpeptide hydrolase and the pebp inhibitor from the archaeon sulfolobus solfataricus.this work describes the identification and characterization of a sulfolobus solfataricus acylpeptide hydrolase, named apeh(ss), recognised as a new protease target of the endogenous pebp inhibitor, sscei. apeh is one of the four members of the prolyl oligopeptidase (pop) family, which removes acylated amino acid residues from the n terminus of oligopeptides. apeh(ss) is a cytosolic homodimeric protein with a molecular mass of 125 kda. it displays a similar exopeptidase and endopeptidase activity ...201020941418
omnipotent role of archaeal elongation factor 1 alpha (ef1α in translational elongation and termination, and quality control of protein synthesis.the molecular mechanisms of translation termination and mrna surveillance in archaea remain unclear. in eukaryotes, erf3 and hbs1, which are homologous to the trna carrier gtpase ef1α, respectively bind erf1 and pelota to decipher stop codons or to facilitate mrna surveillance. however, genome-wide searches of archaea have failed to detect any orthologs to both gtpases. here, we report the crystal structure of arf1 from an archaeon, aeropyrum pernix, and present strong evidence that the authenti ...201020974926
crystallization and preliminary x-ray analysis of a novel dye-linked l-proline dehydrogenase from the aerobic hyperthermophilic archaeon aeropyrum pernix.a novel dye-linked l-proline dehydrogenase from the aerobic hyperthermophilic archaeon aeropyrum pernix was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. the crystals belonged to the tetragonal space group p4(1)2(1)2 or its enantiomorph p4(3)2(1)2, with unit-cell parameters a = b = 61.1, c = 276.3 å, and diffracted to 2.87 å resolution using a cu kα rotating-anode generator with an r-axis vii detector. the asymmetric unit contained ...201021045308
a conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity.in archaea, splicing endonuclease (enda) recognizes and cleaves precursor rnas to remove introns. currently, endas are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. the crenarchaeal heterotetrameric endas can be further classified into two subfamilies based on the size of the structural subunit. subfamily a possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily b possesses a structural subuni ...201021050862
structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase.acylaminoacyl peptidase from aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. the monomer subunit is composed of one hydrolase and one propeller domain. previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. usi ...201021084296
structural and functional insights into aeropyrum pernix oppa, a member of a novel archaeal oppa subfamily.in this study we gain insight into the structural and functional characterization of the aeropyrum pernix oligopeptide-binding protein (oppa(ap)) previously identified from the extracellular medium of an aeropyrum pernix cell culture at late stationary phase. oppa(ap) showed an n-terminal q32 in a pyroglutamate form and c-terminal processing at the level of a threonine-rich region probably involved in protein membrane anchoring. moreover, the oppa(ap) protein released into the medium was identif ...201021097609
crystal structure of the cambialistic superoxide dismutase from aeropyrum pernix k1--insights into the enzyme mechanism and stability.aeropyrum pernix k1, an aerobic hyperthermophilic archaeon, produces a cambialistic superoxide dismutase that is active in the presence of either of mn or fe. the crystal structures of the superoxide dismutase from a. pernix in the apo, mn-bound and fe-bound forms were determined at resolutions of 1.56, 1.35 and 1.48 å, respectively. the overall structure consisted of a compact homotetramer. analytical ultracentrifugation was used to confirm the tetrameric association in solution. in the mn-boun ...201021182595
identify protein-coding genes in the genomes of aeropyrum pernix k1 and chlorobium tepidum tls.the problem that how many protein-coding genes there are in the genome of aeropyrum pernix k1 has confused many scientists since the sequencing in 1999. in this paper, the protein-coding genes in a. pernix k1 are identified from the original and current nite annotation by using the aper_orfs method. consequently, 702 of 704 experimentally validated genes are correctly predicted as coding, which means the sensitivity of the method is 702/704 approximately 99.7%. this sensitivity is one percent hi ...200919108580
deficiency of the trnatyr:psi 35-synthase apus7 in archaea of the sulfolobales order might be rescued by the h/aca srna-guided machinery.up to now, psi formation in trnas was found to be catalysed by stand-alone enzymes. by computational analysis of archaeal genomes we detected putative h/aca srnas, in four sulfolobales species and in aeropyrum pernix, that might guide psi 35 formation in pre-trna(tyr)(gua). this modification is achieved by pus7p in eukarya. the validity of the computational predictions was verified by in vitro reconstitution of h/aca srnps using the identified sulfolobus solfataricus h/aca srna. comparison of pu ...200919139072
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
molecular recognition of tryptophan trna by tryptophanyl-trna synthetase from aeropyrum pernix k1.the identity elements of transfer rna are the molecular basis for recognition by each cognate aminoacyl-trna synthetase. in the archaea system, the tryptophan trna identity has not been determined in detail. to investigate the molecular recognition mechanism of tryptophan trna by tryptophanyl-trna synthetase (trprs) from the hyperthermophilic and aerobic archaeon, aeropyrum pernix k1, various mutant transcripts of tryptophan trna prepared by an in vitro transcription system were examined by over ...200919179361
refolding, characterization and crystal structure of (s)-malate dehydrogenase from the hyperthermophilic archaeon aeropyrum pernix.tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon aeropyrum pernix, and the enzyme was identified as (s)-malate dehydrogenase (mdh), which, when produced in escherichia coli, was mainly obtained as an inactive inclusion body. the inclusion body was dissolved in 6 m guanidine-hcl and gradually refolded to the active enzyme through dilution of the denaturant. the purified recombinant enzyme consisted of four identical subunits with a molecular mass ...200919555779
biophysical studies of the membrane location of the voltage-gated sensors in the hsapbk and kvap k(+) channels.the membrane location of two fragments in two different k(+)-channels, the kvap (from aeropyrum pernix) and the hsapbk (human) corresponding to the putative "paddle" domains, has been investigated by cd, fluorescence and nmr spectroscopy. both domains interact with q = 0.5 phospholipid bicelles, dhpc micelles and with popc vesicles. cd spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. fluorescence quenching studies using soluble acrylamide or ...200919595987
outside the unusual cell wall of the hyperthermophilic archaeon aeropyrum pernix k1.in contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. to identify secreted proteins of the hyperthermophilic archaeon aeropyrum pernix k1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. the experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. because of the lack of signal ...200919640852
gain and loss of an intron in a protein-coding gene in archaea: the case of an archaeal rna pseudouridine synthase gene.we previously found the first examples of splicing of archaeal pre-mrnas for homologs of the eukaryotic cbf5 protein (also known as dyskerin in humans) in aeropyrum pernix, sulfolobus solfataricus, s. tokodaii, and s. acidocaldarirus, and also showed that crenarchaeal species in orders desulfurococcales and sulfolobales, except for hyperthermus butylicus, pyrodictium occultum, pyrolobus fumarii, and ignicoccus islandicus, contain the (putative) cbf5 intron. however, the exact timing of the intro ...200919671140
voltage-dependent conformational changes of kvap s4 segment in bacterial membrane environment.the nature and magnitude of voltage sensor conformational changes during ion channel activation are controversial. we have analyzed the topology of the k(v)ap voltage sensor domain in the absence and presence of a hyperpolarized voltage using native, right-side out membrane vesicles from e. coli. this approach does not disrupt the normal membrane environment of the channel protein and does not involve detergent solubilization. we found that voltage-dependent conformational changes are focused in ...200919713752
purification and characterization of the oxygen-thermostable hydrogenase from the aerobic hyperthermophilic archaeon aeropyrum camini.aeropyrum camini that was isolated from a deep-sea hydrothermal vent chimney, possessed two hydrogenases (161 and 85 kda) in its soluble fraction. the 85-kda hydrogenase was purified to homogeneity using several chromatography columns. the specific activities of the purified hydrogenase were: 14.8 micromol methyl viologen(ox)/mg/min for hydrogen oxidation, and 14.6 micromol methyl viologen(red)/mg/min for proton reduction. the oxygen stabilities of hydrogenases that were purified from a. camini ...200919716518
two complementary enzymes for threonylation of trna in crenarchaeota: crystal structure of aeropyrum pernix threonyl-trna synthetase lacking a cis-editing domain.in protein synthesis, threonyl-trna synthetase (thrrs) must recognize threonine (thr) from the 20 kinds of amino acids and the cognate trna(thr) from different trnas in order to generate thr-trna(thr). in general, an organism possesses one kind of gene corresponding to thrrs. however, it has been recently found that some organisms have two different genes for thrrs in the genome, suggesting that their proteins thrrs-1 and thrrs-2 function separately and complement each other in the threonylation ...200919761773
glu88 in the non-catalytic domain of acylpeptide hydrolase plays dual roles: charge neutralization for enzymatic activity and formation of salt bridge for thermodynamic stability.acylpeptide hydrolase of aeropyrum pernix k1 is composed of a catalytic alpha/beta hydrolase domain and a non-catalytic beta-propeller domain. the glu88 residue of the propeller domain is highly conserved in the prolyl oligopeptidase family and forms an inter-domain salt bridge with arg526, a key residue for substrate binding. we have dissected the functions of glu88 using site-directed mutagenesis, steady-state kinetics analyses, and molecular dynamics simulations. in e88a and e88a/r526k mutant ...200918930847
crystallization and preliminary crystallographic studies of putative threonyl-trna synthetases from aeropyrum pernix and sulfolobus tokodaii.threonyl-trna synthetase (thrrs) plays an essential role in protein synthesis by catalyzing the aminoacylation of trna(thr) and editing misacylation. thrrs generally contains an n-terminal editing domain, a catalytic domain and an anticodon-binding domain. the sequences of the editing domain in thrrss from archaea differ from those in bacteria and eukaryotes. furthermore, several creanarchaea including aeropyrum pernix k1 and sulfolobus tokodaii strain 7 contain two genes encoding either the cat ...200818931432
biochemical characterization of the minichromosome maintenance (mcm) protein of the crenarchaeote aeropyrum pernix and its interactions with the origin recognition complex (orc) proteins.replication in archaea is carried out by proteins that are homologues of eukaryotic counterparts. however, the archaeal systems tend to be much simpler with fewer different genes encoding the core functions than in eukaryotic counterparts. in many archaea, there is a single minichromosome maintenance (mcm) homologue, presumed to be the replicative helicase and between one and three origin recognition complex (orc) homologues involved in binding to the replication origins. here we describe the cl ...200819053250
structures of two archaeal diphthine synthases: insights into the post-translational modification of elongation factor 2.the target of diphtheria toxin is the diphthamide residue in translation elongation factor 2 (ef-2), which is generated by a three-step post-translational modification of a specific histidine residue in the ef-2 precursor. in the second modification step, an s-adenosylmethionine-dependent methyltransferase, diphthine synthase (ds), catalyzes the trimethylation of the ef-2 precursor. the homodimeric crystal structures of the archaeal diphthine synthases from pyrococcus horikoshii ot3 and aeropyru ...200818391406
oxidation of archaeal peroxiredoxin involves a hypervalent sulfur intermediate.the oxidation of thiol groups in proteins is a common event in biochemical processes involving disulfide bond formation and in response to an increased level of reactive oxygen species. it has been widely accepted that the oxidation of a cysteine side chain is initiated by the formation of cysteine sulfenic acid (cys-soh). here, we demonstrate a mechanism of thiol oxidation through a hypervalent sulfur intermediate by presenting crystallographic evidence from an archaeal peroxiredoxin (prx), the ...200818436649
cell cycle characteristics of crenarchaeota: unity among diversity.the hyperthermophilic archaea acidianus hospitalis, aeropyrum pernix, pyrobaculum aerophilum, pyrobaculum calidifontis, and sulfolobus tokodaii representing three different orders in the phylum crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. the overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generati ...200818502873
the genomics of luca.to understand the nature and evolution of luca, or last universal common ancestor, the minimum genome of luca has been identified based on the genes common to the eight primitive euryarchaea and crenarchaea species methanopyrus kandleri, methanothermobacter thermautotrophicum, methanococcus jannaschii, pyrococcus abyssi, pyrococcus furiosus, pyrococcus horikoshii, aeropyrum pernix and pyrobaculum aerophilum, together with the methanogenesis genes of the primitive methanogens. the 424 protein enc ...200818508609
the conserved n-terminal helix of acylpeptide hydrolase from archaeon aeropyrum pernix k1 is important for its hyperthermophilic activity.the acylpeptide hydrolases from hyperthermophilic archaeon aeropyrum pernix k1 has a short conserved n-terminal helix in its family. the role of this n-terminal helix in the function of the hyperthermophilic enzyme, however, is unknown. here, we investigated this question by protein engineering and biophysical methods. we found that a mutant (deltan21) with the n-terminal helix deleted is no longer functional at the optimum temperature for wt enzyme (95 degrees c), required for the survival of a ...200818590836
the microbial ecology of a high-temperature near-neutral spring situated in rotorua, new zealand.a combination of both culture and culture-independent techniques were used to investigate the microbial ecology of a near-neutral, high-temperature hot spring (designated aq1) in rotorua, new zealand. the active microbial members of the community were targeted by analyzing biofilms that developed on surfaces incubated in situ in aq1. colonization of surfaces was rapid as indicated by atp assay and microscopic observation. dna-based analysis of both colonized surfaces and pool water from aq1 reve ...200817207984
crystal structure of an archaeal specific dna-binding protein (ape10b2) from aeropyrum pernix k1.dna binding proteins are essential in all organisms, and they play important roles in both compacting and regulating the genetic material. all thermophilic and hyperthermophilic archaea encode one or more copies of alba or sso10b, which is a small, abundant, basic protein that binds dna. here, we present the crystal structure of ape10b2 from aeropyrum pernix k1 at 1.70 a. although the overall structure resembles the known alba protein fold, a significant conformational change was observed in the ...200818004791
biochemical characterization of dna-binding proteins from pyrobaculum aerophilum and aeropyrum pernix.several representatives of the crenarchaeal branch of the archaea contain highly abundant, small, positively charged proteins exemplified by the sso7d protein from sulfolobus solfataricus. these proteins bind to dna in a non-sequence-specific manner. using publicly available genomic sequence information, we identified a second class of small crenarchaeal dna-binding proteins represented by the pyrobaculum aerophilum open reading frame 3192-encoded (pae3192) protein and its paralogs. we investiga ...200818064401
a unique catalytic triad revealed by the crystal structure of ape0912, a short-chain dehydrogenase/reductase family protein from aeropyrum pernix k1. 200818175326
structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.it is widely accepted that the catalytic activity of serine proteases depends primarily on the asp-his-ser catalytic triad and other residues within the vicinity of this motif. some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. in acylaminoacyl peptidase from the thermophile aeropyrum pernix, the main chain nh group of gly369 is one of the hydrogen bond donors forming the oxyanion binding site ...200818325786
peroxiredoxins as cellular guardians in sulfolobus solfataricus: characterization of bcp1, bcp3 and bcp4.peroxiredoxins are ubiquitous enzymes that are part of the oxidative stress defense system. in the present study, we identified three peroxiredoxins [bacterioferritin comigratory protein (bcp)1, bcp3 and bcp4] in the genome of the aerobic hyperthermophilic archaeon sulfolobus solfataricus. based on the cysteine residues conserved in the deduced aminoacidic sequence, bcp1 and bcp4 can be classified as 2-cys peroxiredoxins and bcp3 as a 1-cys peroxiredoxin. a comparative study of the recombinant b ...200818355320
in silico analysis of voltage-gated sodium channel in relation to ddt resistance in vector mosquitoes.the voltage-gated sodium channel (vgsc) is the target site for insecticides such as ddt and synthetic pyrethroids. a single base (a-t) change in the knock-down resistance (kdr) allele leads to an amino acid substitution at position 267 that confers the target-mediated resistance to ddt and synthetic pyrethroids in anopheles gambiae. a theoretical model of the vgsc domain ii that contains the site of mutation was constructed using the k;+ channel protein of aeropyrum pernix as a template. the val ...200718391234
in vivo characterization of thermal stabilities of aeropyrum pernix cellular components by differential scanning calorimetry.revival studies of aeropyrum pernix show that the viability of cells and cell recovery after heat treatment depends on the temperature of treatment. differential scanning calorimetry (dsc) is used to analyze the relative thermal stabilities of cellular components of a. pernix and to identify the cellular components responsible for the observed lag phase and reduced maximum growth following a heat treatment. dsc thermograms show 5 visible endothermic transitions with 2 major transitions. dsc anal ...200718026224
determination of phenylalanine trna recognition sites by phenylalanyl-trna synthetase from hyperthermophilic archaeon, aeropyrum pernix k1.phenylalanine trna identity has been determined in the bacteria and the eukaryote system, but remains unknown for the archaea system. to investigate the molecular recognition mechanism of phenylalanine trna by phenylalanyl-trna synthetase from hyperthermophilic and aerobic archaeon, aeropyrum pernix k1, various mutant transcripts of phenylalanine trna prepared by an in vitro transcription system were examined by overexpressed a. pernix phenylalanyl trna synthetase. the results indicated that ant ...200718029739
the phosphofructokinase-b (mj0406) from methanocaldococcus jannaschii represents a nucleoside kinase with a broad substrate specificity.recently, unusual non-regulated atp-dependent 6-phosphofructokinases (pfk) that belong to the pfk-b family have been described for the hyperthermophilic archaea desulfurococcus amylolyticus and aeropyrum pernix. putative homologues were found in genomes of several archaea including the hyperthermophilic archaeon methanocaldococcus jannaschii. in this organism, open reading frame mj0406 had been annotated as a pfk-b sugar kinase. the gene encoding mj0406 was cloned and functionally expressed in e ...200717021658
cc1, a novel crenarchaeal dna binding protein.the genomes of the related crenarchaea pyrobaculum aerophilum and thermoproteus tenax lack any obvious gene encoding a single-stranded dna binding protein (ssb). ssbs are essential for dna replication, recombination, and repair and are found in all other genomes across the three domains of life. these two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the alba protein), while most other archaea have at least two different abundant chromatin proteins. we perfo ...200717085561
properties of the alpha subunit of a chaperonin from the hyperthermophilic crenarchaeon aeropyrum pernix k1.the gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon aeropyrum pernix k1 (apcpna) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. the gene (accession no. ape0907) from a. pernix k1 showed some homology with other group ii chaperonins from archaea. the recombinant apcpna protein has a molecular mass of 60 kda, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited atpase activity with ...200717092293
cloning and expression of a dna ligase from the hyperthermophilic archaeon staphylothermus marinus and properties of the enzyme.the gene encoding staphylothermus marinus dna ligase (sma dna ligase) was cloned and sequenced. the gene contains an open reading frame consisting of 1836bp, which encodes for 611 amino acid residues. upon alignment of the entire amino acid sequence, sma dna ligase showed a high degree of sequence homology with the hyperthemophilic archaeal dna ligases, 67% identity with aeropyrum pernix k1, and 40% identity with both pyrococcus abyssi and thermococcus kodakarensis. an extremely high sequence id ...200717118474
thermal stability and biochemical properties of isocitrate dehydrogenase from the thermoacidophilic archaeon thermoplasma acidophilum.isocitrate dehydrogenase [idh; ec 1.1.1.42] from the thermoacidophilic archaeon thermoplasma acidophilum (taidh) showed high thermal stability with an apparent melting temperature, t(m), of 82.2 and 84.5 degrees c at ph 7.5 and 5.8, respectively. based on structural alignment of taidh with idh from aeropyrum pernix (apidh) and archaeoglobus fulgidus (afidh) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in apidh and afidh were identified in the ...200717123127
characterization of thermostable native alkaline phosphatase from an aerobic hyperthermophilic archaeon, aeropyrum pernix k1.this paper reports the characterization of an alkaline phosphatase (ap) from an aerobic hyperthermophilic archaeon aeropyrum pernix k1. the native ap was purified into homogeneity. the enzyme is predicted as a homodimeric structure with a native molecular mass of about 75 kda and monomer of about 40 kda. apparent optimum ph and temperature were estimated at 10.0 and above 95 degrees c, respectively. magnesium ion increased both the stability and the activity of the enzyme. a. pernix ap has been ...200717256119
crystallization of the archaeal transcription termination factor nusa: a significant decrease in twinning under microgravity conditions.the transcription termination factor nusa from aeropyrum pernix was crystallized using a counter-diffusion technique in both terrestrial and microgravity environments. crystallization under microgravity conditions significantly reduced the twinning content (1.0%) compared with terrestrially grown crystals (18.3%) and improved the maximum resolution from 3.0 to 2.29 a, with similar unit-cell parameters. based on a comparison of the crystal parameters, the effect of microgravity on protein crystal ...200717277442
crystal structure and rna-binding analysis of the archaeal transcription factor nusa.the transcription factor nusa functions in transcriptional regulation involving termination in bacteria. a nusa homolog consisting of only the two kh domains is widely conserved in archaea, but its function remains unknown. we have found that aeropyrum pernix nusa strongly binds to a certain cu-rich sequence near a termination signal. our crystal structure of a. pernix nusa revealed that its spatial arrangement is quite similar to that of the kh domains of bacterial nusa. thus, we consider archa ...200717288993
the acylaminoacyl peptidase from aeropyrum pernix k1 thought to be an exopeptidase displays endopeptidase activity.mammalian acylaminoacyl peptidase, a member of the prolyl oligopeptidase family of serine peptidases, is an exopeptidase, which removes acylated amino acid residues from the n terminus of oligopeptides. we have investigated the kinetics and inhibitor binding of the orthologous acylaminoacyl peptidase from the thermophile aeropyrum pernix k1 (apaap). complex ph-rate profiles were found with charged substrates, indicating a strong electrostatic effect in the surroundings of the active site. unexpe ...200717350041
genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate.di-myo-inositol 1,1'-phosphate (dip) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. although the dip biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. in this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipa and dipb) that remained missing. in thermotoga marit ...200717360515
extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes.in all three domains of life, dna replication begins at specialized loci termed replication origins. in bacteria, replication initiates from a single, clearly defined site. in contrast, eukaryotic organisms exploit a multitude of replication origins, dividing their genomes into an array of short contiguous units. recently, the multiple replication origin paradigm has also been demonstrated within the archaeal domain of life, with the discovery that the hyperthermophilic archaeon sulfolobus has t ...200717392430
phylogenomic analysis of proteins that are distinctive of archaea and its main subgroups and the origin of methanogenesis.the archaea are highly diverse in terms of their physiology, metabolism and ecology. presently, very few molecular characteristics are known that are uniquely shared by either all archaea or the different main groups within archaea. the evolutionary relationships among different groups within the euryarchaeota branch are also not clearly understood.200717394648
thermal stability of isocitrate dehydrogenase from archaeoglobus fulgidus studied by crystal structure analysis and engineering of chimers.isocitrate dehydrogenase from archaeoglobus fulgidus (afidh) has an apparent melting temperature (t(m)) of 98.5 degrees c. to identify the structural features involved in thermal stabilization of afidh, the structure was solved to 2.5 a resolution. afidh was strikingly similar to mesophilic idh from escherichia coli (ecidh) and displayed almost the same number of ion pairs and ionic networks. however, two unique inter-domain networks were present in afidh; one three-membered ionic network betwee ...200717401542
a single amino acid substitution in the dna-binding domain of aeropyrum pernix dna ligase impairs its interaction with proliferating cell nuclear antigen.proliferating cell nuclear antigen (pcna) is known as a dna sliding clamp that acts as a platform for the assembly of enzymes involved in dna replication and repair. previously, it was reported that a crenarchaeal pcna formed a heterotrimeric structure, and that each pcna subunit has distinct binding specificity to pcna-binding proteins. here we describe the pcna-binding properties of a dna ligase from the hyperthermophilic crenarchaeon aeropyrum pernix k1. based on our findings on the pyrococcu ...200717487442
specific interactions of three proliferating cell nuclear antigens with replication-related proteins in aeropyrum pernix.proliferating cell nuclear antigen (pcna) is a well-known multifunctional protein involved in eukaryotic and archaeal dna transactions. the homotrimeric pcna ring encircles double-stranded dna within its central hole and tethers many proteins on dna. plural genes encoding pcna-like proteins have been found in the genome sequence of crenarchaeal organisms. we describe here the biochemical properties of the three pcnas, pcna1, pcna2 and pcna3, from the hyperthermophilic archaeon, aeropyrum pernix. ...200717493121
analysis of synonymous codon usage in aeropyrum pernix k1 and other crenarchaeota microorganisms.in this study, a comparative analysis of the codon usage bias was performed in aeropyrum pernix k1 and two other phylogenetically related crenarchaeota microorganisms (i.e., pyrobaculum aerophilum str. im2 and sulfolobus acidocaldarius dsm 639). the results indicated that the synonymous codon usage in a. pernix k1 was less biased, which was highly correlated with the gc(3s) value. the codon usage patterns were phylogenetically conserved among these crenarchaeota microorganisms. comparatively, it ...200717498625
bifunctional ctp:inositol-1-phosphate cytidylyltransferase/cdp-inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper)thermophiles.the pathway for the synthesis of di-myo-inositol-phosphate (dip) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (nmr) (n. borges, l. g. gonçalves, m. v. rodrigues, f. siopa, r. ventura, c. maycock, p. lamosa, and h. santos, j. bacteriol. 188:8128-8135, 2006). here, a genomic approach was used to identify the genes involved in the synthesis of dip. ...200717526717
glyceraldehyde-3-phosphate ferredoxin oxidoreductase (gapor) and nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (gapn), key enzymes of the respective modified embden-meyerhof pathways in the hyperthermophilic crenarchaeota pyrobaculum aerophilum and aeropyrum pernix.the growth of pyrobaculum aerophilum on yeast extract and nitrate was stimulated by the addition of maltose. extracts of maltose/yeast extract/nitrate-grown cells contained all enzyme activities of a modified embden-meyerhof (em) pathway, including atp-dependent glucokinase, phosphoglucose isomerase, atp-dependent 6-phosphofructokinase, fructose-1,6-phosphate aldolase, triose-phosphate isomerase, gapor, phosphoglycerate mutase, enolase and pyruvate kinase. the activity of gapor was stimulated ab ...200717559573
temperature- and ph-induced structural changes in the membrane of the hyperthermophilic archaeon aeropyrum pernix k1.the influence of ph and temperature on the structural organization, fluidity and permeability of the hyperthermophilic archaeon membrane was investigated in situ by a combination of electron paramagnetic resonance (epr) and fluorescence emission spectroscopy. for epr measurements, aeropyrum pernix cells, after growing at different phs, were spin-labeled with the doxyl derivative of palmitic acid methylester (mefasl(10,3)). from the epr spectra maximal hyperfine splitting (2a (max)) and empirical ...200717713807
structural biology. getting dna to unwind. 200717761872
structural basis of dna replication origin recognition by an orc protein.dna replication in archaea and in eukaryotes share many similarities. we report the structure of an archaeal origin recognition complex protein, orc1, bound to an origin recognition box, a dna sequence that is found in multiple copies at replication origins. dna binding is mediated principally by a c-terminal winged helix domain that inserts deeply into the major and minor grooves, widening them both. however, additional dna contacts are made with the n-terminal aaa+ domain, which inserts into t ...200717761880
polyphasic evidence delineating the root of life and roots of biological domains.twenty different lines of polyphasic evidence obtained from trna and protein sequences, anticodon usages, gene contents, metabolism and geochemistry have made possible the identification of a last universal common ancestor (luca) phylogenetically located proximal to the hyperthermophilic methanogenic archaeon methanopyrus. combined with analysis of high-similarity cross-domain trna pairs, the evidence also suggests a thermotoga-proximal last bacterial common ancestor (lbaca) that originated from ...200717884304
proteome analysis of an aerobic hyperthermophilic crenarchaeon, aeropyrum pernix k1.we analyzed the proteome of a crenararchaeon, aeropyrum pernix k1, by using the following four methods: (i) two-dimensional page followed by maldi-tof ms, (ii) one-dimensional sds-page in combination with two-dimensional lc-ms/ms, (iii) multidimensional lc-ms/ms, and (iv) two-dimensional page followed by amino-terminal amino acid sequencing. these methods were found to be complementary to each other, and biases in the data obtained in one method could largely be compensated by the data obtained ...200616455681
cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon pyrococcus abyssi.four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea pyrococcus abyssi, pyrococcus horikoshii, pyrococcus furiosus, and aeropyrum pernix (growth temperature 90-100 degrees c). the nitrilase encoding genes were cloned and overexpressed in escherichia coli. enzymatic activity could only be detected in the case of py. abyssi. this recombinant nitrilase was purified by heat treatment of e. coli crude extract followed by anion-exchange ...200616495079
crystal structures of tyrosyl-trna synthetases from archaea.tyrosyl-trna synthetase (tyrrs) catalyzes the tyrosylation of trna(tyr) in a two-step reaction. tyrrs has the "high" and "kmsks" motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and atp. here, we determined the crystal structures of archaeoglobus fulgidus and pyrococcus horikoshii tyrrss in the l-tyrosine-bound form at 1.8a and 2.2a resolutions, respectively, and that of aeropyrum pernix tyrrs in the substrate-free form at 2.2 a. the conformation of the ...200616325203
the intrinsic flexibility of the kv voltage sensor and its implications for channel gating.analysis of the crystal structures of the intact voltage-sensitive potassium channel kvap (from aeropyrum pernix) and kv1.2 (from rat brain), along with the isolated voltage sensor (vs) domain from kvap, raises the question of the exact nature of the voltage-sensing conformational change that triggers activation of kv and related voltage-gated channels. molecular dynamics simulations of the isolated vs of kvap in a detergent micelle environment at two different temperatures (300 k and 368 k) hav ...200616326912
optimization of recombinant hyperthermophilic esterase production from agricultural waste using response surface methodology.the aim of this work was to evaluate the capability of corn steep liquor being a low cost substrate on the recombinant protein production by cultivating recombinant escherichia coli and increasing the production of hyperthermophilic esterase (he). the effect of corn steep liquor, mineral salt and trace metals on hyperthermophilic esterase production was investigated by means of a five-level three-factor central composite rotatable design. optimized values of the factors were determined and a max ...200616330210
crystal structure of thioredoxin peroxidase from aerobic hyperthermophilic archaeon aeropyrum pernix k1. 200616342268
geomicrobiological exploration and characterization of a novel deep-sea hydrothermal system at the toto caldera in the mariana volcanic arc.novel hydrothermal activities accompanying effluent white smokers and elemental sulfur chimney structures at the north-east lava dome of the toto caldera depression in the mariana volcanic arc have been explored and characterized by geochemical and microbiological surveys. white smoker hydrothermal fluids were observed in the potential hydrothermal activity centre of the field and represented the maximal temperature of 170 degrees c and the lowest ph of 1.6. the chimney structures, all consistin ...200616343320
external k+ modulates the activity of the arabidopsis potassium channel skor via an unusual mechanism.plant outward-rectifying k+ channels mediate k+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (k). intriguingly, the gating of these channels depends on the extracellular k+ concentration, although the ions carrying the current are derived from inside the cell. this k+ dependence confers a sensitivity to the extracellular k+ concentration ([k+]) that ensures that the channels mediate k+ efflux only, regardless of the [k+ ...200616623889
identification of the first archaeal oligopeptide-binding protein from the hyperthermophile aeropyrum pernix.the archaeon aeropyrum pernix grows optimally at 90 degrees c and derives energy primarily from aerobic degradation of complex proteinaceous substrates. the ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent atp-binding cassette (abc) transporters. this study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (oppa(ap)) from the extr ...200616636888
overexpression of the genes from thermophiles in escherichia coli by high-temperature cultivation.twenty-nine aminotransferase genes from pyrococcus horikoshii, aeropyrum pernix, and sulfolobus tokodaii were cloned and expressed in escherichia coli. the expression of several of the genes at 15, 25, or 37 degrees c resulted in the formation of insoluble protein aggregates. therefore, we developed a simple method to express these genes into soluble proteins, by cultivating e. coli clones at a higher temperature. thus, four genes could be expressed efficiently into soluble and active enzymes by ...200616652221
discrimination of esterase and peptidase activities of acylaminoacyl peptidase from hyperthermophilic aeropyrum pernix k1 by a single mutation.it has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. using saturation mutagenesis, we investigated the role of a conserved residue (arg(526)) at the active site of acylaminoacyl peptidase from hyperthermophilic aeropyrum pernix k1 in substrate discrimination and catalytic mechanism. this enzyme has both peptidase and esterase activities. the esterase activity of the wild-t ...200616670095
cysteine biosynthesis in trichomonas vaginalis involves cysteine synthase utilizing o-phosphoserine.trichomonas vaginalis is an early divergent eukaryote with many unusual biochemical features. it is an anaerobic protozoan parasite of humans that is thought to rely heavily on cysteine as a major redox buffer, because it lacks glutathione. we report here that for synthesis of cysteine from sulfide, t. vaginalis relies upon cysteine synthase. the enzyme (tvcs1) can use either o-acetylserine or o-phosphoserine as substrates. the k(m) values of the enzyme for sulfide are very low (0.02 mm), sugges ...200616735516
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