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1.
Cellobiose uptake in the hyperthermophilic archaeon Pyrococcus furiosus is mediated by an inducible, high-affinity ABC transporter
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The hyperthermophilic archaeon Pyrococcus furiosus can utilize different beta-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K(m) = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosus membranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtA gene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters. 相似文献
2.
Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus 总被引:2,自引:0,他引:2
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Kil YV Baitin DM Masui R Bonch-Osmolovskaya EA Kuramitsu S Lanzov VA 《Journal of bacteriology》2000,182(1):130-134
The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C). 相似文献
3.
Babu E Kanai Y Chairoungdua A Kim DK Iribe Y Tangtrongsup S Jutabha P Li Y Ahmed N Sakamoto S Anzai N Nagamori S Endou H 《The Journal of biological chemistry》2003,278(44):43838-43845
A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters. 相似文献
4.
5.
Porcelli M Concilio L Peluso I Marabotti A Facchiano A Cacciapuoti G 《The FEBS journal》2008,275(8):1900-1914
We report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The gene SSO0505 encoding SsCU-NH was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The alignment of the amino acid sequences of SsCU-NH with nucleoside hydrolases whose 3D structures have been solved indicates that the amino acid residues involved in the calcium- and ribose-binding sites are preserved. SsCU-NH is highly thermophilic with an optimum temperature of 100 degrees C and is characterized by extreme thermodynamic stability (T(m) = 106 degrees C) and kinetic stability (100% residual activity after 1 h incubation at 90 degrees C). Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. The structure of the enzyme determined by homology modeling provides insight into the proteolytic analyses as well as into mechanisms of thermal stability. This is the first nucleoside hydrolase from Archaea. 相似文献
6.
G Greller R Horlacher J DiRuggiero W Boos 《The Journal of biological chemistry》1999,274(29):20259-20264
We report the cloning, sequencing, and expression of malK encoding the ATP-hydrolyzing subunit of the maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis. According to the deduced amino acid sequence, MalK consists of 372 amino acids with a calculated molecular weight of 41,787. It shows 47% identity with the MalK protein of Escherichia coli and high sequence conservation in important regions. C-terminal His-tagged MalK was purified. The soluble protein appeared monomeric by molecular sieve chromatography and showed ATPase activity. Enzymatic activity was highest at 80 degrees C with a Km of 150 microM and a Vmax of 0.55 micromol of ATP hydrolyzed/min/mg of protein. ADP was not a substrate but a competitive inhibitor (Ki 230 microM). GTP and CTP were also hydrolyzed. ATPase activity was inhibited by N-ethylmaleimide but not by vanadate. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the ABC transporters in these two phylogenetic branches. 相似文献
7.
The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400 mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe. 相似文献
8.
Molecular and functional characterization of a family of amino acid transporters from Arabidopsis 总被引:1,自引:0,他引:1
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More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter. 相似文献
9.
Cloning and characterization of the Halobacillus trueperi betH gene, encoding the transport system for the compatible solute glycine betaine 总被引:2,自引:0,他引:2
Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity. A fragment of the glycine betaine transporter betH gene was obtained from the genome of H. trueperi with degenerate primers. Through Southern blot hybridization and inverse PCR, a 5.1 kb EcoRI fragment containing the complete betH gene was identified and subsequently sequenced. The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembrane regions. BetH showed 56% identity to the OpuD of Bacillus subtilis which belongs to the betaine/carnitine/choline transporter (BCCT) family. Its putative promoter region was highly homologous to sigmaB-dependent promoter of B. subtilis. A 2.6 kb fragment containing the betH gene was cloned into pUC18 and transformed into the Escherichia coli MKH13. The accumulation of glycine betaine in transformed E. coli MKH13 bacteria was confirmed using 13C nuclear magnetic resonance spectroscopy. 相似文献
10.
Tang Guiliang Zhu-Shimoni Judith X. Amir Rachel Ben-Tzvi Zchori Inbal Galili Gad 《Plant molecular biology》1997,34(2):287-294
As in many bacterial species, the first enzymatic reaction of the aspartate-family pathway in plants is mediated by several isozymes of aspartate kinase (AK) that are subject to feedback inhibition by the end-product amino acids lysine or threonine. So far, only cDNAs and genes encoding threonine-sensitive AKs have been cloned from plants. These were all shown to encode polypeptides containing two linked activities, namely AK and homoserine dehydrogenase (HSD), similar to the Escherichia coli thrA gene encoding a threonine-sensitive bifunctional AK/HSD isozyme. In the present report, we describe the cloning of a new Arabidopsis thaliana cDNA that is relatively highly homologous to the E. coli lysC gene encoding the lysine-sensitive AK isozyme. Moreover, similar to the bacterial lysine-sensitive AK, the polypeptide encoded by the present cDNA is monofunctional and does not contain an HSD domain. These observations imply that our cloned cDNA encodes a lysine-sensitive AK. Southern blot hybridization detected a single gene highly homologous to the present cDNA, plus an additional much less homologous gene. This was confirmed by the independent cloning of an additional Arabidopsis cDNA encoding a lysine-sensitive AK (see accompanying paper). Northern blot analysis suggested that the gene encoding this monofunctional AK cDNA is abundantly expressed in most if not all tissues of Arabidopsis. 相似文献
11.
Molecular structure of a novel cholesterol-responsive A subclass ABC transporter,ABCA9 总被引:2,自引:0,他引:2
Piehler A Kaminski WE Wenzel JJ Langmann T Schmitz G 《Biochemical and biophysical research communications》2002,295(2):408-416
We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis. 相似文献
12.
We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV, which was slightly higher than that of Trx from E. coli (-270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria. 相似文献
13.
Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD. 总被引:4,自引:3,他引:1
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The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis. 相似文献
14.
利用PCR扩增技术从极端嗜热古菌Pyrococcus horikoshii中得到预测为几丁二糖脱乙酰酶的基因(Dacph,PH0499),将其克隆入表达质粒pET15b,并在E.coliBL21_codonPlus(DE3)_RIL中表达获得可溶的Dacph重组蛋白(31.6kDa),TLC分析证明Dacph能够脱去N_乙酰氨基葡萄糖及几丁二糖的一个乙酰基,并与氨基葡萄糖苷酶(BglAPh)共同作用水解几丁二糖生成氨基葡萄糖,从而被命名为一种几丁二糖脱乙酰酶。与Pyrococcus horikoshii中外切氨基葡萄糖苷酶等共同作用,Dacph可能在嗜热球古菌独特的几丁质降解途径中起重要作用。 相似文献
15.
16.
Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A 总被引:19,自引:0,他引:19
Sugawara M Nakanishi T Fei YJ Huang W Ganapathy ME Leibach FH Ganapathy V 《The Journal of biological chemistry》2000,275(22):16473-16477
We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states. 相似文献
17.
In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1. 总被引:1,自引:0,他引:1
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The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit. 相似文献
18.
H Bahl G Burchhardt A Spreinat K Haeckel A Wienecke B Schmidt G Antranikian 《Applied and environmental microbiology》1991,57(5):1554-1559
The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains. 相似文献
19.
alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases. 总被引:5,自引:1,他引:4
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H Bahl G Burchhardt A Spreinat K Haeckel A Wienecke B Schmidt G Antranikian 《Applied microbiology》1991,57(5):1554-1559
The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains. 相似文献
20.
The trimethylammonium compound glycine betaine (N,N, N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus sigmaB-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed Km value of 7.9 microM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL+ parent, signifying the role of the transporter in Listeria osmotolerance. 相似文献