Biochemical diversity among sulfur-dependent, hyperthermophilic microorganisms

Abstract: Hyperthermophiles are a recently discovered group of microorganisms that grow at and above 90°C. They currently comprise over 20 different genera, and except for two novel bacteria, all are classified as Archaea. The majority of these organisms are obligately anaerobic heterotrophs that reduce elemental sulfur (S°) to H₂S. The best studied from a biochemical perspective are the archaeon, Pyrococcus furiosus , and the bacterium, Thermotoga maritima , both of which are saccharolytic. P. furiosus is thought to contain a new type of Entner-Doudoroff pathway for the conversion of carbohydrates ultimately to acetate, H₂ and CO₂. The pathway is independent of nicotinamide nucleotides and involves novel types of ferredoxin-linked oxidoreductases, one of which has tungsten, a rarely used element, as a prosthetic group. The only site of energy conservation is at the level of acetyl CoA, which in the presence of ADP and phosphate is converted to acetate and ATP in a single step. In contrast, T. maritima utilizes a conventional Embden-Meyerhof pathway for sugar oxidation. P. furiosus also utilizes peptides as a sole carbon and energy source. Amino acid oxidation is thought to involve glutamate dehydrogenase together with at least three types of novel ferredoxin-linked oxidoreductases which catalyze the oxidation of 2-ketoglutarate, aryl pyruvates and formaldehyde. One of these enzymes also utilizes tungsten. In P. furiosus , virtually all of the reductant that is generated during the catabolism of both carbohydrates and peptides is channeled to a cytoplasmic hydrogenase. This enzyme is now termed sulhydrogenase, as it reduces both protons to H₂ and S°(or polysulfide) to H₂S. S° reduction appears to lead to the conservation of energy in P. furiosus but not in T. maritima , although the mechanism by which this occurs is not known.     

超耐热酸性α-淀粉酶基因在多型汉逊酵母中表达及与在毕赤酵母中表达的比较研究

利用毕赤酵母的质粒载体pPIC9K将极端耐热古菌Pyrococcusfuriosus的超耐热酸性α-淀粉酶(Amy)基因转化到多型汉逊酵母HP-6中,获得重组汉逊酵母。经过甲醇进行诱导,表达产物的酶活性检测和SDS-PAGE电泳,证明α-淀粉酶(Amy)在多型汉逊酵母中利用AOX1启动子和α-因子信号肽有效表达并分泌到胞外。该酶的最适反应温度为90～100℃,最适作用pH为4.0～5.0,较之重组毕赤酵母的最适作用pH还低0.5。此外与毕赤酵母的重组蛋白相比,重组汉逊酵母α-淀粉酶不仅菌株筛选简便、周期短,而且具有更容易筛选到高拷贝转化子以及适用于大规模工业发酵等优点。     

火球菌8-氧鸟嘌呤DNA糖苷酶基因克隆、表达纯化和酶学性质研究

【目的】在大肠杆菌中表达火球菌8-氧鸟嘌呤DNA糖苷酶,纯化得到重组火球菌8-氧鸟嘌呤DNA糖苷酶,在此基础上系统研究火球菌8-氧鸟嘌呤DNA糖苷酶的酶学特征。【方法】构建8-氧鸟嘌呤DNA糖苷酶重组表达质粒,将重组质粒转化Escherichia coli Rosetta(DE3),利用IPTG诱导表达重组蛋白,通过Ni2+亲和层析柱纯化重组蛋白;最后利用含8-氧鸟嘌呤损伤的寡核苷酸作为底物,测定8-氧鸟嘌呤DNA糖苷酶的酶学性质。【结果】在大肠杆菌中成功诱导表达了重组火球菌8-氧鸟嘌呤DNA糖苷酶,经Ni2+亲和纯化后蛋白纯度大于95%。在体外鉴定了重组火球菌8-氧鸟嘌呤DNA糖苷酶的酶学性质。结果表明重组火球菌8-氧鸟嘌呤DNA糖苷酶可以切除DNA中的8-氧鸟嘌呤(8-Oxo-G,GO)损伤碱基,并且具有AP裂解酶活性。重组火球菌8-氧鸟嘌呤DNA糖苷酶催化反应的最适pH值和温度分别是pH 8.5和55°C。除Zn2+对火球菌8-氧鸟嘌呤DNA糖苷酶的酶促反应有明显的抑制作用外,实验中测定的其它二价离子(Mn2+,Mg2+,Ca2+,Ni2+,Co2+,Cu2+)对其没有明显的影响。离子强度在50-100 mmol/L范围内对其酶促反应影响不大,超过100 mmol/L时有明显的抑制作用。与8-氧鸟嘌呤互补的碱基差异对火球菌8-氧鸟嘌呤DNA糖苷酶切除8-氧鸟嘌呤损伤的效率影响不大;但与单链DNA相比,双链DNA是优选底物,切割效率如下:GO/C≈GO/G≈GO/T≈GO/AGO/-。【结论】在大肠杆菌中成功表达,并Ni2+亲和纯化了火球菌8-氧鸟嘌呤DNA糖苷酶,生化研究表明制备的重组蛋白具有8-氧鸟嘌呤DNA糖苷酶活性,可能负责切除火球菌基因组DNA中的8-氧鸟嘌呤损伤。     

Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.     

Membrane proteins with molecular masses of 88, 90 and 150 kDa are responsible for binding of human immunoglobulin G Fc fragment to the native cells of Mycoplasma salivarium

Abstract Growth of Pyrococcus furiosus was studied in batch cultures with cellobiose, maltose and pyruvate as limiting substrates. Fermentation mass balances of all conversions were complete. These data suggested that the pathway for conversion of the disaccharides is essentially the same. The molar growth yields on maltose and cellobiose were about equal (±50 g cell dry weight (cdw) per mol disaccharide). The molar growth yield on pyruvate was ±6.3 g cdw mol⁻¹. Growth yields were influenced by the hydrogen partial pressure. When P. furiosus was co-cultured with a methanogen a yield of 56 g cdw mol⁻¹ disaccharide and 8.6 g cdw mol⁻¹ pyruvate was obtained. When the data were interpreted according to the proposed pyroglycolytic pathway, the calculated Y_ATP values were different for the various pH₂ conditions, suggesting that an additional energy conserving site may be present in the pathway.     

Equilibrium shifts in enzyme reactions at high pressure

The effect of pressure on the equilibrium of a reaction was studied. Theoretical equilibrium constants and product concentrations have been calculated at elevated pressures. The theory is illustrated with an example of l-malate synthesis catalyzed by a fumarase. To study shifts in the equilibrium relatively low pressures can be applied (50–200 MPa), but our calculations show that for process optimisation much higher pressures (up to 1000 MPa) have to be used.

At these higher pressures, more stable enzymes are needed. We performed experiments with the hyperthermophilic β-glycosidase from Pyrococcus furiosus as a catalyst. Oligosaccharides were synthesized from glucose in an equilibrium reaction at pressures from 0.1 to 500 MPa. The enzyme remained active at 500 MPa. The equilibrium of the reaction was influenced by pressure and shifted towards the hydrolysis side, decreasing final oligosaccharide concentrations with increasing pressure. This pressure dependence of the final product concentration and the equilibrium constant could be described with a positive reaction volume of 2.4 mol/cm³.     

Sugar utilization and anoxia tolerance in rice roots acclimated by hypoxic pretreatment

Although most cereal roots cannot elongate under anoxic conditions, primary roots of three-day-old rice (Oryza sativa L.) seedlings were able to elongate during a 24-h period of anoxia. Hypoxic pretreatment (H-PT) increased the elongation of their roots. Sucrose synthase (EC 2.4.1.13), glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4), pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) activities were increased by anoxia in both H-PT and non-pretreated (N-PT) roots. However, these activities were greater in the H-PT roots than in the N-PT roots. The average rate of production of ethanol for the initial 6h after the onset of anoxia was 3.7 and 1.4 micromolg(-1) fresh weight h(-1) for the H-PT and N-PT roots, respectively, suggesting that ethanolic fermentation may increase more quickly in the H-PT roots than in the N-PT roots. Roots of the seedlings lost ATP and total adenine nucleotides in anoxia, however, the H-PT roots maintained higher levels of ATP and total adenine nucleotides compared to the N-PT roots. These results show that rice roots are able to utilize the set of enzymes involved in the metabolism of soluble sugars under anoxia. The ability to maintain an active fermentative metabolism for production of ATP by fueling the glycolytic pathway with fermentable carbohydrate is probably greater in H-PT than in N-PT roots.     

Overexpression of an archaeal protein in yeast: secretion bottleneck at the ER

Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging. Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. To determine whether a eukaryotic host, S. cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase's secretion and activity. We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a transformant capable of secreting approximately 10 mg/L in batch culture. All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck. Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck. Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association. Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields.     

Molecular characterization of phosphoglycerate mutase in archaea

The interconversion of 3-phosphoglycerate and 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). In bacteria and eukaryotes two structurally distinct enzymes have been found, a cofactor-dependent and a cofactor-independent (iPGM) type. Sequence analysis of archaeal genomes did not find PGMs of either kind, but identified a new family of proteins, distantly related to iPGMs. In this study, these predicted archaeal PGMs from Pyrococcus furiosus and Methanococcus jannaschii have been functionally produced in Escherichia coli, and characterization of the purified proteins has confirmed that they are iPGMs. Analysis of the available microbial genomes indicates that this new type of iPGM is widely distributed among archaea and also encoded in several bacteria. In addition, as has been demonstrated in certain bacteria, some archaea appear to possess an alternative, cofactor-dependent PGM.     

超耐热酸性α-淀粉酶基因的克隆及其在酵母细胞中的表达

用PCR方法扩增来源于极端嗜热厌氧古菌Pyrococcus furiosus中的超耐热酸性α-淀粉酶的结构基因,将该结构基因引入载体pPIC9K中,将重组质粒pPIC9K-Amy转化大肠杆菌DH5α细胞,测序结果表明,克隆到的α-淀粉酶结构基因为1305bp,其编码的成熟肽为435个氨基酸。将正确构建的重组质粒转化毕赤酵母GS115细胞,得到酵母工程菌株。在酵母α-Factor及AOX1基因启动子和终止信号的调控下,超耐热酸性α-淀粉酶在甲醇酵母中大量表达并分泌到胞外,该酶的表达受甲醇的严格调控和诱导,随着诱导培养时间的增加,在培养基上清液中的单位体积酶活力相应上升,在诱导培养7d后酶活力达到最大值。该酶最适反应温度为90~100℃,最适反应pH值为4.5~5.5。该酶具有非常好的温度稳定性,在100℃条件下热处理5h,仍具有60%以上的酶活力。该酶的这些优点使其非常适于在工业生产上应用。     

Conservation from rat to human of cytosolic phosphoenolpyruvate carboxykinase and the control of its gene expression

Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.     

Straight-chain fatty alcohols in the hyperthermophilic archaeon Pyrococcus furiosus

Two straight-chain fatty alcohols (n-hexadecanol and n-octadecanol) were found in the neutral lipid fraction extracted from Pyrococcus furiosus cells. They were identified by thin-layer and gas-liquid chromatography, mass and infrared spectra, and chemical modification. The fatty alcohols accounted for 54% of the neutral lipid of the cell. Received: March 8, 2000 / Accepted: May 8, 2000     

Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase

In the first step of this study, type 2 isopentenyl diphosphate isomerase (IDI2) from Pyrococcus furiosus (pf-IDI2), a hyperthermophilic microorganism, was cloned and overexpressed in E. coli. After purification, hyperthermophilic behavior of this protein was approached by means of enzymatic assays and thermal denaturation studies. Compared with the mesophilic Streptococcus pneumoniae IDI2, which unfolds and looses activity above 50 degrees C, pf-IDI2 is still folded and active at 80 degrees C. Molecular modeling was applied, in a parallel step, to understand the molecular basis of thermal stability. Comparison of IDI2 from S. pneumoniae, T. thermophilus, and P. furiosus suggested that additional charged residues present in the hyperthermophilic enzyme might contribute to its higher thermal stability. This could increase the number of salt bridges between monomers of IDI2 in P. furiosus enzyme and, hence, decrease flexibility of loops or N-terminal segment, thereby enhancing its thermal stability.     

Structure determination of a new protein from backbone-centered NMR data and NMR-assisted structure prediction

Targeting of proteins for structure determination in structural genomic programs often includes the use of threading and fold recognition methods to exclude proteins belonging to well-populated fold families, but such methods can still fail to recognize preexisting folds. The authors illustrate here a method in which limited amounts of structural data are used to improve an initial homology search and the data are subsequently used to produce a structure by data-constrained refinement of an identified structural template. The data used are primarily NMR-based residual dipolar couplings, but they also include additional chemical shift and backbone-nuclear Overhauser effect data. Using this methodology, a backbone structure was efficiently produced for a 10 kDa protein (PF1455) from Pyrococcus furiosus. Its relationship to existing structures and its probable function are discussed.     

Maillard reactions and increased enzyme inactivation during oligosaccharide synthesis by a hyperthermophilic glycosidase

The thermostable Pyrococcus furiosus beta-glycosidase was used for oligosaccharide production from lactose in a kinetically controlled reaction. Our experiments showed that higher temperatures are beneficial for the absolute as well as relative oligosaccharide yield. However, at reaction temperatures of 80 degrees C and higher, the inactivation rate of the enzyme in the presence of sugars was increased by a factor of 2 compared to the inactivation rate in the absence of sugars. This increased enzyme inactivation was caused by the occurrence of Maillard reactions between the sugar and the enzyme. The browning of our reaction mixture due to Maillard reactions was modeled by a cascade of a zeroth- and first-order reaction and related to enzyme inactivation. From these results we conclude that modification of only a small number of amino groups already gives complete inactivation of the enzyme.