极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2 、Fe3 、Mn2 等金属离子对酶活性有微弱抑制作用。     

A non-radioactive assay for selenophosphate synthetase activity using recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-¹⁴C] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The K_M and k_cat for Sephs2-Sec60Cys were determined to be 26 μM and 0.352 min^?1, respectively.     

Expression,purification and characterization of recombinant protein tyrosine phosphatase from Thermus thermophilus HB27

The low-molecular-weight protein tyrosine phospha- tases （PTPase） exist ubiquitously in prokaryotes and eukaryotes and play important roles in the regulation of physiological activities. We report here the expression, purification and characterization of an active and soluble PTPase from Thermus thermophilus HB27 in Escherichia coli. This PTPase has an optimum pH range of 2.8-4.8 when using p-nitrophenyl phosphate as the substrate. The thermal inactivation results indicate a high thermal stability of this enzyme, with the optimum temperature of 75℃ for activity. It can be activated by Mn^2＋, Mg^2＋, Ca^2＋, Ba^2＋, and Ni^2＋, but inhibited by Zn^2＋, Cu^2＋, Cl^-, and SO^2-. These results suggest that this heat-resistant PTPase may play important roles in vivo in the adaptation of the microorganism to extreme temperatures and specific nutritional conditions.     

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.     

Crystal structure of ribosomal protein L27 from Thermus thermophilus HB8

Ribosomal protein L27 is located near the peptidyltransferase center at the interface of ribosomal subunits, and is important for ribosomal assembly and function. We report the crystal structure of ribosomal protein L27 from Thermus thermophilus HB8, which was determined by the multiwavelength anomalous dispersion method and refined to an R-factor of 19.7% (R(free) = 23.6%) at 2.8 A resolution. The overall fold is an all beta-sheet hybrid. It consists of two sets of four-stranded beta-sheets formed around a well-defined hydrophobic core, with a highly positive charge on the protein surface. The structure of ribosomal protein L27 from T. thermophilus HB8 in the RNA-free form is investigated, and its functional roles in the ribosomal subunit are discussed.     

嗜热菌HB8 Fe-S簇生物合成相关酶SufS与SufE重组酶的体外复合体功能

Fe-S簇在细胞的生物学过程中发挥重要作用,参与电子传递和基因调节等过程. 以嗜热菌HB8基因组为模板,通过PCR扩增获得Fe-S簇SUF系统生物合成途径中的sufS与sufE两个基因. 将目的基因分别连接到表达载体pET28a和pGEX-6P-1上,构建重组质粒sufS-pET28a和sufE-pGEX-6P-1. 重组质粒转化E.coli BL21(DE3)菌株进行蛋白质表达及亲和层析纯化. 研究证明,SufS具有脱硫酶活性,而SufE的存在可增强其活性. 体外重组实验表明,在60 ℃孵育SufS和SufE 1.0～2.5 h,其复合体脱硫酶活性约是单体SufS的2倍,但两者相互作用时间短暂. 若通过交联剂Mts Atf Biotin使SufS与SufE共价连接,此时SufSE酶活性是SufS的6倍左右,并可持续72 h.     

Solution structure of ribosomal protein L16 from Thermus thermophilus HB8

Ribosomal protein L16 is an essential component of the bacterial ribosome. It organizes the architecture of aminoacyl tRNA binding site in the ribosome 50S subunit. The three-dimensional structure of L16 from Thermus thermophilus HB8 was determined by NMR. In solution, L16 forms an alpha+beta sandwich structure combined with two additional beta sheets located at the loop regions connecting the two layers. The terminal regions and a central loop region did not show any specific secondary structure. The structured part of L16 could be superimposed well on the C(alpha) model of L16 determined in the crystal structure of the ribosome 50S subunit. By overlaying the L16 solution structure onto the coordinates of the ribosome crystal structure, we constructed the combined model that represents the ribosome-bound state of L16 in the detailed structure. The model showed that L16 possesses residues in contact with helices 38, 39, 42, 43 and 89 of 23S rRNA and helix 4 of 5S rRNA. This suggests its broad effect on the ribosome architecture. Comparison of L16 with the L10e protein, which is the archaeal counterpart, showed that they share a common fold, but differ in some regions of functional importance, especially in the N-terminal region. All known mutation sites in L16 that confer resistance to avilamycin and evernimicin were positioned so that their side-chains were exposed to solvent in the internal cavity of the ribosome. This suggests the direct participation of L16 as a part of the binding site for antibiotics.     

Thermus thermophilus HB8葡萄糖异构酶在大肠杆菌中表达

为了增加高热稳定性的葡萄糖异构酶的得率,采用PCR技术扩增得到Thermus thermophilusHB8葡萄糖异构酶基因xylA,连接到表达载体pET-22b( )上,获得重组质粒pET-22b( )-xylA。将重组质粒转化到大肠杆菌Rosetta(DE3)中,经IPTG诱导后,通过半胱氨酸-咔唑法测葡萄糖异构酶酶活。重组菌经诱导培养,SDS-PAGE电泳结果显示出明显的分子量约为44 kD特异性蛋白质条带,比酶活约为18.562 U/mg,比野生型菌株提高了2倍。     

Crystal structure of a lysine biosynthesis enzyme,LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.     

Denaturation studies by fluorescence and quenching of thermophilic protein NAD+-glutamate dehydrogenase from Thermus thermophilus HB8

Fluorescence techniques have been used to study the structural characteristics of many proteins. The thermophilic enzyme NAD-glutamate dehydrogenase from Thermus thermophilus HB8 is found to be a hexameric enzyme. Fluorescence spectra of native and denatured protein and effect of denaturants as urea and guanidine hydrochloride on enzyme activity of thermophilic glutamate dehydrogenase (t-GDH) have been analyzed. Native t-GDH presents the maximum emission at 338 nm. The denaturation process is accompanied by an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum. Fluorescence quenching by external quenchers, KI and acrylamide, has also been carried out.     

Crystallization and preliminary X-ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides.

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 A. Based on the assumption of one 32-kDa monomer per asymmetric unit, the Vm value is 2.83 A3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 A resolution.     

Crystallization and preliminary crystallographic analysis of ribosomal protein S8 from Thermus thermophilus

Crystals have been obtained for recombinant ribosomal protein S8 from Thermus thermophilus produced by Escherichia coli. The protein crystals have been grown in 40 mM potassium phosphate buffer (pH 6.0) in hanging drops equilibrated against saturated ammonium sulfate (unbuffered) with 2-methyl-2,4-pentandiol (v/v). The crystals belong to the space group P4₁₍₃₎2₁2 with cell parameters a= b= 67.65 Å, c= 171.12 Å. They diffract x-rays to 2.9 Å resolution. © 1997 Wiley-Liss, Inc.     

Crystal structure of the conserved protein TT1542 from Thermus thermophilus HB8

The TT1542 protein from Thermus thermophilus HB8 is annotated as a conserved hypothetical protein, and belongs to the DUF158 family in the Pfam database. A BLAST search revealed that homologs of TT1542 are present in a wide range of organisms. The TT1542 homologs in eukaryotes, PIG-L in mammals, and GPI12 in yeast and protozoa, have N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase activity. Although most of the homologs in prokaryotes are hypothetical and have no known function, Rv1082 and Rv1170 from Mycobacterium tuberculosis are enzymes involved in the mycothiol detoxification pathway. Here we report the crystal structure of the TT1542 protein at 2.0 A resolution, which represents the first structure for this superfamily of proteins. The structure of the TT1542 monomer consists of a twisted beta-sheet composed of six parallel beta-strands and one antiparallel beta-strand (with the strand order 3-2-1-4-5-7-6) sandwiched between six alpha-helices. The N-terminal five beta-strands and four alpha-helices form an incomplete Rossmann fold-like structure. The structure shares some similarity to the sugar-processing enzymes with Rossmann fold-like domains, especially those of the GPGTF (glycogen phosphorylase/glycosyl transferase) superfamily, and also to the NAD(P)-binding Rossmann fold domains. TT1542 is a homohexamer in the crystal and in solution, the six monomers forming a cylindrical structure. Putative active sites are suggested by the structure and conserved amino acid residues.     

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.     

Cocrystallization of Lysyl–tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys

Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNA^lYS or Escherichia coli tRNA^lys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18–22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 Å, c = 119 Å) and diffract to at least 2.7 Å resolution. However the homocomplex is of space group P42₁2 and the heterocomplex of space group I422. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.     

Crystallization and preliminary X-ray crystallographic studies on Type III antifreeze protein.

Type III antifreeze protein, more specifically the recombinant QAE-Sephadex-binding isoform, has been crystallized in 50-55% saturated ammonium sulfate, 0.1 M sodium acetate, pH 4.0-4.5. The resultant crystals belong to the orthorhombic space group P212121 with a = 32.60 A, b = 39.00 A, and c = 46.57 A and diffract to at least 1.7 A. A set of 1.7-A native data has been collected, with completeness 93.4% and Rsym of 0.069. Initial screening for heavy-atom derivatives has yielded a Pt-bound derivative.     

A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8

Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.