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.     

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.     

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.     

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.     

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     

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg⁻¹. The enzyme required Mg²⁺ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K _m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V _max of 243 U mg⁻¹) and 1.47 mM (apparent V _max of 197 U mg⁻¹), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na⁺ and K⁺. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K _m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V _max of 2.9 U mg⁻¹) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases. Received: August 14, 1998 / Accepted: December 2, 1998     

The Entner-Doudoroff pathway: history, physiology and molecular biology

Abstract The Entner-Doudoroff pathway is now known to be very widely distributed in nature. Biochemical and physiological studies show that the Entner-Doudoroff pathway can operate in a linear and catabolic mode, in a 'cyclic' mode, in a modified mode involving non-phosphorylated intermediates, or in alternative modes involving C₁ metabolism and anabolism. Molecular and genetic analyses of the Entner-Doudoroff pathway in Zymomonas mobilis, Escherichia coli and Pseudomonas aeruginosa have led to an improved understanding of some fundamental aspects of metabolic controls. It can be argued that the Entner-Doudoroff pathway is more primitive than Embden-Meyerhof-Parnas glycolysis.     

非编码RNA及其研究进展

目前 ,大致有 10类ncRNA的神秘面纱被揭开 ,这些分布广泛的ncRNA ,在多种生物体的重要生命过程中具有不可替代的作用。它们的长度变化很大 ,但都不编码蛋白质 ,直接在RNA水平上发挥着调控作用 ,如细胞的生长和分化 ,个体的发育时序调控 ,以及疾病的形成和抑制等。因而 ,对ncRNA的深入研究将对基因功能开发、人类疾病防治及生物进化的探索有重要意义。     

Overview on sugar metabolism and its control in Lactococcus lactis - the input from in vivo NMR

The wide application of lactic acid bacteria in the production of fermented foods depends to a great extent on the unique features of sugar metabolism in these organisms. The relative metabolic simplicity and the availability of genetic tools made Lactococcus lactis the organism of choice to gain insight into metabolic and regulatory networks. In vivo nuclear magnetic resonance has proven a very useful technique to monitor non-invasively the dynamics of intracellular metabolite and co-factor pools following a glucose pulse. Examples of the application of this methodology to identify metabolic bottlenecks and regulatory sites are presented. The use of this information to direct metabolic engineering strategies is illustrated.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP = 0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.     

Decorin gene expression and its regulation in human keratinocytes

In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.     

hsBLyS基因在中国仓鼠卵巢细胞中的表达及其在免疫小鼠中诱发的抗体反应

为建立稳定表达人可溶性B淋巴细胞刺激因子(hsBLyS)的中国仓鼠卵巢(CHO)细胞系,从人胎盘cDNA文库中扩增hsBLyS基因,将人红细胞生成素(EPO)信号肽序列和hsBLyS基因重叠延伸为融合基因;融合基因分别插入pcDNA3、pcDNA3.1、pEFneo质粒;磷酸钙共沉淀将表达质粒和标志质粒pSVdhfr,共转染CHOdhfr细胞;选择培养液筛选,经氨甲喋呤选择压力扩增表达,获稳定表达hsBLyS的细胞系,表达量由0.13μg/ml上升至0.55μg/ml;同时利用pEFneo/hsBLyS重组质粒免疫BALB/c小鼠,检测结果表明小鼠产生hsBLyS的特异性抗体。本实验建立了稳定表达hsBLyS的CHO细胞系,对其基因免疫小鼠抗体产生的特点做了初步探讨,为hsBLyS进一步研究奠定了良好的基础