Purification and characterization of YihA,an essential GTP-binding protein from Escherichia coli

YihA has previously been characterized as an essential gene of unknown function in both Escherichia coli and Bacillus subtilis. It is conserved in bacteria and represents an attractive target for the discovery of new antibiotics. YihA encodes a putative GTP-binding protein. We have cloned and overexpressed the gene encoding E. coli YihA and initiated biochemical studies as a first step towards understanding its biological function. We showed by circular dichroism that the purified protein has a secondary structure typical of most GTP-binding proteins. It binds guanine nucleotides specifically, as demonstrated by fluorescence resonance energy transfer between 2'-(or-3')-O-(N-methylanthraniloyl) nucleotides (mant-nucleotides) and the tryptophans of YihA. The K(d) values for GDP and GTP were determined by competition with 2'-(or-3')-O-(N-methylanthraniloyl) GDP to be 3 and 27 microM, respectively. Using mutants of YihA we show that nucleotide binding occurs at the putative GTP-binding domain predicted from the primary sequence.     

Characterization of GTPase activity of TrmE, a member of a novel GTPase superfamily, from Thermotoga maritima

A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium. T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. K(m) and k(cat) at 70 degrees C were 833 microM and 9.3 min(-1), respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.     

Purification, characterization and crystallization of ERA, an essential GTPase from Escherichia coli

ERA is an essential GTPase widely conserved in bacteria. Homologues of ERA are also present in higher eukaryotic cells. ERA is involved in bacterial cell cycle control at a point preceding cell division. In order to aid the functional investigation of ERA and to facilitate structure-function studies, we have undertaken the X-ray crystallographic analysis of this protein. Here, we report the purification and crystallization procedures and results. The purified ERA exhibits nucleotide-binding activity and GTP-hydrolytic activity. ERA is one of the very few multi-domain GTPases crystallized to date.     

Subunit exchange by CheA histidine kinases from the mesophile Escherichia coli and the thermophile Thermotoga maritima

Dimerization of the chemotaxis histidine kinase CheA is required for intersubunit autophosphorylation [Swanson, R. V., Bourret, R. B., and Simon, M. I. (1993) Mol. Microbiol. 8, 435-441]. Here we show that CheA dimers exchange subunits by the rate-limiting dissociation of a central four-helix bundle association domain (P3), despite the high stability of P3 versus unfolding. P3 alone determines the stability and exchange properties of the CheA dimer. For CheA proteins from the mesophile Escherichia coli and the thermophile Thermotoga maritima, subunit dissociation activates at temperatures where the respective organisms live (37 and 80 degrees C). Under destabilizing conditions, P3 dimer dissociation is cooperative with unfolding. Chemical denaturation is reversible for both EP3 and TP3. Aggregation accompanies thermal unfolding for both proteins under most conditions, but thermal unfolding is reversible and two-state for EP3 at low protein concentrations. Residue differences within interhelical loops may account for the contrasted thermodynamic properties of structurally similar EP3 and TP3 (41% sequence identity). Under stabilizing conditions, greater correlation between activation energy for dimer dissociation and P3 stability suggests more unfolding in the dissociation of EP3 than TP3. Furthermore, destabilization of extended conformations by glycerol slows relative dissociation rates more for EP3 than for TP3. Nevertheless, at physiological temperatures, neither protein likely unfolds completely during subunit exchange. EP3 and TP3 will not exchange subunits with each other. The receptor coupling protein CheW reduces the subunit dissociation rate of the T. maritima CheA dimer by interacting with the regulatory domain P5.     

Pleiotropic changes resulting from depletion of Era, an essential GTP-binding protein in Escherichia coli

Phenotypic analysis of a temperature-sensitive era mutant strain indicates that Escherichia coli cells depleted of Era undergo many physiological changes. At 43 degrees C, a completely non-permissive temperature, growth is arrested because of loss of the gene and depletion of the Era protein. Depletion of Era at 43 degrees C results in depressed synthesis of heat-shock proteins DnaK, GroEL/ES, D33.4 and C62.5, lack of thermal induction of ppGpp pool levels, and increased capacity for carbon source metabolism through the citric acid cycle. Thus, in addition to inhibition of cell growth and viability, loss of Era function results in pleiotropic changes including abnormal adaptation to thermal stress.     

海栖热袍菌极耐高温木聚糖酶基因xynB64在大肠杆菌中的融合表达

来源于极端嗜热菌海栖热袍菌(Thermotoga maritima MSB8)的木聚糖酶B具有极高的热稳定性,在饲料、造纸、能源和食品医药行业具有巨大应用潜力。携带酶基因xynB64的pET28a(+)重组载体在宿主大肠杆菌BL21(DE3)中诱导表达,重组酶活力较低。更换宿主为携带稀有tRNA基因的大肠杆菌:BL21-CodonPlus(DE3)-RIPL和Rosetta(DE3)后,酶活力分别提高了197%和277%,但是后者中的表达会形成部分包涵体。宿主菌为大肠杆菌Rosetta(DE3),更换载体为4种融合表达载体pET32a(+)、pET42a(+)、pET43.1a(+)和pMAL-c2X进行表达,重组酶分别融合了Trx、GST、Nus和MBP标签。其中Rosetta(DE3)/pMAL-c2X-xynB64表达酶活力最高,相当于Rosetta(DE3)/pET28a-xynB64表达酶的88%,而且目的酶表达量占全细胞蛋白的40%,几乎不形成包涵体。     

极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达

以海栖热袍菌 (Thermotoga maritima) MSB8菌株基因组DNA为模板,通过PCR扩增出木聚糖酶（XylanaseB）基因, 将此基因克隆至大肠杆菌表达载体pET_28a（+）和毕赤酵母表达载体pPIC9K,并分别转化大肠杆菌 BL21和毕赤酵母GS115。该木聚糖酶在大肠杆菌细胞中表达量高, 但不能分泌; 而在毕赤酵母细胞的表达产物可分泌至胞外。酶学性质分析表明,此酶分子量约为40kD,其最适反应温度为90℃, 最适反应pH值为6.65,且在碱性条件下稳定,具有重要的工业应用前景。     

Characterization of the autophosphorylation of Era, an essential Escherichia coli GTPase

Era is an essential protein in Escherichia coli which binds both GTP and GDP and has an intrinsic GTPase activity. Studies on the role of GTP/GDP binding and GTPase activity in an attempt to understand its function lead to the observation that Era is autophosphorylated. The autophosphorylated reaction is specific for GTP and cannot use ATP as a phosphoryl group donor. The reaction velocity is of first order with respect to protein concentration, suggesting an intramolecular mechanism. Autophosphorylation occurs at serine and threonine residues. The major phosphorylated tryptic peptide isolated after autophosphorylation has been identified as ISITSR, from residue 33 to 38. The peptide contains the site of phosphorylation and two potential sites for serine and threonine phosphorylation. Subsequently, both the threonine residue at position 36 and the serine residue at position 37 were altered to alanine. The double mutant Era, but not individual single mutants, was unable to functionally complement the growth of an E. coli strain which cannot produce wild-type Era protein at high temperature. This suggests that either threonine 36 or serine 37 has to exist for the function of Era In vivo. phosphorylation of Era was also examined by two-dimensional gel electrophoresis. Era has been previously assigned two distinct positions having two different X-Y co-ordinates: one of the spots (H032.0) was identified as phosphorylated Era, indicating that a substantial portion of Era in the cell is indeed phosphorylated. Therefore, Era autophosphorylation is likely to play an important physiological role in the cell. The sequence encoding the C-terminus previously published had a missing C between A900 and GgO1. As a resuit of the frameshift, Era consists of 301 residues, 15 fewer than originaiiy reported.     

A thermostable 5-enolpyruvylshikimate-3-phosphate synthase from Thermotoga maritima enhances glyphosate tolerance in Escherichia coli and transgenic Arabidopsis

Extremophiles - 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) overexpression, attempting to provide excess EPSPS to combine with glyphosate, is one way to improve glyphosate resistance of...     

极耐热性阿拉伯糖苷酶基因的表达、纯化及酶学性质研究

采用PCR从海栖热袍菌(Thermotoga maritima)克隆出编码极耐热稳定性阿拉伯糖苷酶基因,以pET20b为表达质粒,与其C末端6个组氨酸标签序列融合,在大肠杆菌中得到高效表达。基因表达产物通过热处理和亲和层析柱纯化后,酶纯度达电泳均一。纯化重组酶稳定性检测表明,阿拉伯糖苷酶活性最适作用温度和最适作用pH分别为90～95℃和pH 5.0～5.5,在pH 4.2～8.2之间酶活力稳定,95℃的半衰期为4h;SDSPAGE测得酶的分子量为56.57 kD,与理论推算值相吻合。在所测定的底物中,阿拉伯糖苷酶仅对对硝基苯阿拉伯呋喃糖苷（pNPAF）有专一性水解作用,其动力学参数Km值为018mmol/L, Vmax为139μmol/min·mg。     

极耐热性b-葡萄糖醛酸酶的高效表达和酶学性质及其 应用

从海栖热袍菌克隆出编码热稳定性b-葡萄糖醛酸酶基因, 以热激载体pHsh为表达质粒, 在大肠杆菌中得到高效表达。基因表达产物通过一步热处理后, 酶纯度达电泳均一。纯化重组酶酶学性质研究表明, b-葡萄糖醛酸酶的最适反应温度为80oC, 最适反应pH为5.0, pH 5.8~ 8.2之间酶的稳定性较好, 80oC的半衰期为2 h, SDS-PAGE结果显示分子量为65.9 kD, 与理论推算值相吻合。以对硝基苯-b-葡萄糖醛酸苷(pNPG)为底物时, 其动力学参数Km值0.18 mmol/L, Vmax值为312 u/mg。初步的应用分析表明, 该重组酶能催化甘草酸转化为甘草次酸。     

Deficiency of essential GTP-binding protein ObgE in Escherichia coli inhibits chromosome partition

GTP-binding proteins are involved in cell proliferation, development, signal transduction, protein elongation, etc. and construct the GTPase superfamily, whose structures and sequence motifs (G-1 to G-5) are highly conserved from prokaryote to eukaryote. Obg of Bacillus subtilis and Obg homologues of other bacteria belong to the GTPase superfamily and have been suggested as being essential for cell growth, development and monitoring of intracellular levels of GTP. We identified the Obg homologue in Escherichia coli, a protein previously known as YhbZ, which we have renamed ObgE. Double cross-over experiments showed that the obgE gene is essential for growth in E. coli. From characterization of the obgE temperature-sensitive mutant, we found that DNA replication was not inhibited, that the nucleoids did not partition and instead remained in the middle of cell, and that the cells elongated. Overproduction of ObgE also resulted in aberrant chromosome segregation. These data suggested that ObgE is involved directly or indirectly in E. coli chromosome partitioning. Characterization studies showed that ObgE is abundant in normal cells, partially associated with the membrane and does not associate with ribosomes such as in Obg of B. subtilis. We purified ObgE protein from a cell extract of E. coli, and the purified ObgE had GTPase activity and DNA-binding ability.     

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli.

The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb HindIII DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50,088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590,000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.     

The [Fe-Fe]-hydrogenase maturation protein HydF from Thermotoga maritima is a GTPase with an iron-sulfur cluster

The active site of [Fe-Fe]-hydrogenases is composed of a di-iron complex, where the two metal atoms are bridged together by a putative di(thiomethyl)amine molecule and are also ligated by di-nuclear ligands, namely carbon monoxide and cyanide. Biosynthesis of this metal site is thought to require specific protein machinery coded by the hydE, hydF, and hydG genes. The HydF protein has been cloned from the thermophilic organism Thermotoga maritima, purified, and characterized. The enzyme possesses specific amino acid signatures for GTP binding and is able to hydrolyze GTP. The anaerobically reconstituted TmHydF protein binds a [4Fe-4S] cluster with peculiar EPR characteristics: an S = 1/2 signal presenting a high field shifted g-value together with a S = 3/2 signal, similar to those observed for [4Fe-4S] clusters ligated by only three cysteines. HYSCORE spectroscopy experiments were carried out to determine the nature of the fourth ligand of the cluster, and its exchangeability was demonstrated with the formation of a [4Fe-4S]-imidazole complex.     

MazG,a nucleoside triphosphate pyrophosphohydrolase,interacts with Era,an essential GTPase in Escherichia coli

Era is an essential GTPase in Escherichia coli, and Era has been implicated in a number of cellular functions. Homologues of Era have been identified in various bacteria and some eukaryotes. Using the era gene as bait in the yeast two-hybrid system to screen E. coli genomic libraries, we discovered that Era interacts with MazG, a protein of unknown function which is highly conserved among bacteria. The direct interaction between Era and MazG was also confirmed in vitro, being stronger in the presence of GDP than in the presence of GTPgammaS. MazG was characterized as a nucleoside triphosphate pyrophosphohydrolase which can hydrolyze all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PP(i), with a preference for deoxynucleotides. A mazG deletion strain of E. coli was constructed by replacing the mazG gene with a kanamycin resistance gene. Unlike mutT, a gene for another conserved nucleotide triphosphate pyrophosphohydrolase that functions as a mutator gene, the mazG deletion did not result in a mutator phenotype in E. coli.     

An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli

EmrE is an Escherichia coli 12-kDa protein that confers resistance to toxic compounds, by actively removing them in exchange with protons. The protein includes eight charged residues. Seven of these residues are located in the hydrophilic loops and can be replaced with either Cys or another amino acid bearing the same charge, without impairing transport activity. Glu-14 is the only charged residue in the membrane domain and is conserved in all the proteins of the family. We show here that this residue is the site of action of dicyclohexylcarbodiimide, a carbodiimide known to act in hydrophobic environments. When Glu-14 was replaced with either Cys or Asp, resistance was abolished. Whereas the E14C mutant displays no transport activity, the E14D protein shows efflux and exchange at rates about 30-50% that of the wild type. The maximal DeltapH-driven uptake rate of E14D is only 10% that of the wild type. The mutant shows a different pH profile in all the transport modes. Our results support the notion that Glu-14 is an essential part of a binding domain shared by substrates and protons but mutually exclusive in time. This notion provides the molecular basis for the obligatory exchange catalyzed by EmrE.     

Exopolygalacturonate lyase from Thermotoga maritima: cloning,characterization and organic synthesis application

A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.     

SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima

Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.