Gene expression and molecular characterization of a thermostable trehalose phosphorylase fromThermoanaerobacter tengcongensis

A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-glucose-1-phosphate (α-Glc-1-P) and glucose, was cloned fromThermoanaerobacter tengcongensis and successfully expressed inEscherichia coli. The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70°C (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual activity after incubation at 50°C for 7 h. Under the optimum reaction conditions, 50 μg crude enzyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-Glc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L trehalose is degraded within the same time period. Dot blotting revealed that thetreP gene inT. tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of theT. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for industrial production of trehalose.     

Cloning and characterization of a gene encoding trehalose phosphorylase (TP) from Pleurotus sajor-caju

Complementary DNA for a gene encoding trehalose phosphorylase (TP) that reversibly catalyzes trehalose synthesis and degradation from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose was cloned from Pleurotus sajor-caju. The cDNA of P. sajor-caju TP (designated PsTP, GenBank Accession No. AF149777) encodes a polypeptide of 751 amino acids with a deduced molecular mass of 83.7 kDa. The PsTP gene is expressed in mycelia, pilei, and stipes of fruiting bodies. Trehalose phosphorylase PsTP was purified from PsTP-transformed Escherichia coli. The enzyme catalyzes both the phosphorolysis of trehalose to produce alpha-Glc-1-P and glucose, and the synthesis of trehalose. The apparent K(m) values for trehalose and Pi in phosphorolytic reaction at pH 7.0 were 74.8 and 5.4 mM, respectively. The PsTP gene complemented Saccharomyces cerevisiae Deltatps1, Deltatps2 double-mutant cells, allowing their growth on glucose medium. Furthermore, yeast transformed with PsTP produced 2-2.5-fold more trehalose than non-transformants or cells transformed with empty vector only.     

A novel trehalose synthesizing pathway in the hyperthermophilic Crenarchaeon <Emphasis Type="Italic">Thermoproteus tenax</Emphasis>: the unidirectional TreT pathway

In the genome of the hyperthermophilic archaeon Thermoproteus tenax a gene (treS/P) encoding a protein with similarity to annotated trehalose phosphorylase (TreP), trehalose synthase (TreS) and more recently characterized trehalose glycosyltransferring synthase (TreT) was identified. The treS/P gene as well as an upstream located ORF of unknown function (orfY) were cloned, heterologously expressed in E. coli and purified. The enzymatic characterization of the putative TreS/P revealed TreT activity. However, contrary to the previously characterized reversible TreT from Thermococcus litoralis and Pyrococcus horikoshii, the T. tenax enzyme is unidirectional and catalyzes only the formation of trehalose from UDP (ADP)-glucose and glucose. The T. tenax enzyme differs from the reversible TreT of T. litoralis by its preference for UDP-glucose as co-substrate. Phylogenetic and comparative gene context analyses reveal a conserved organization of the unidirectional TreT and OrfY gene cluster that is present in many Archaea and a few Bacteria. In contrast, the reversible TreT pathway seems to be restricted to only a few archaeal (e.g. Thermococcales) and bacterial (Thermotogales) members. Here we present a new pathway exclusively involved in trehalose synthesis--the unidirectional TreT pathway--and discuss its physiological role as well as its phylogenetic distribution.     

Purification and properties of α-glucose 1-phosphate-forming trehalose phosphorylase from a basidiomycete,Pleurotus ostreatus

Pleurotus ostreatus produced a high activity of α-glucose 1-phosphate (α-Glc 1-P) forming trehalose phosphorylase in vegetative mycelia and fruit-bodies. The enzyme was purified to homogeneity from the fruit-bodies by a procedure involving ammonium sulfate fractionation, DEAE-cellulose column chromatographies and cellulose phosphate column chromatographies. The enzyme catalyzes both the phosphorolysis of trehalose to produce α-Glc 1-P and glucose, and the synthesis of trehalose. It was not active toward other α- or β-glucosyl disaccharides and polysaccharides. The optimum pH was 7.0 for phosphorolysis and 6.4 for synthesis of trehalose. The Km values for trehalose and Pi in phospholytic reaction were 75 mM and 4.2 mM, respectively. Those for glucose and α-Glc 1-P in synthetic reaction were 505 mM and 38 mM, respectively. The estimated molecular mass by the sedimentation equilibrium method using an ultracentrifuge was 120 kDa. The molecular mass of the subunit (61 kDa) by SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a dimer of two identical subunits. The addition of glycerol higher than 25% into the enzyme solution stabilized its activity. The removal of phosphorus ions from the enzyme solution, by means of dialysis or electrophoresis, caused inactivation of the enzyme, probably by dissociation of the holoenzyme into the subunit proteins.     

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30°C. The optimum pH ranges for trehalose degradation and synthesis were 6.0–7.5 and 6.0–7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P_i (K_i=2.0 mM). The enzyme was highly specific towards trehalose, P_i, glucose and α-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P_i, glucose and α-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K_m values were 61, 4.7, 24 and 6.3 mM for trehalose, P_i, glucose and α-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.     

一个新的高温产氢菌及产氢特性的研究

利用Hungate滚管技术从西藏山南地区热泉淤泥中分离到一株高温产氢的厌氧发酵细菌T42。菌株T42革兰氏染色反应为阴性,但KOH裂解试验证实其为革兰氏阳性杆菌。菌体大小为0.7μm~0.9μm×3.2μm~7μm,不运动,不产芽孢。其生长温度范围为32℃~69℃,最适生长温度为60℃~62℃,生长pH范围为5.0~8.8,最适生长pH为7.0~7.5,代时30min。有机氮源是T42菌株的必需生长因子。菌株T42利用淀粉、纤维二糖、蔗糖、麦芽糖、糊精、果糖、糖原和海藻糖等底物生长并发酵产氢,发酵葡萄糖的终产物为乙酸、乙醇、H2和CO2。G C含量为31.2mol%。系统发育分析表明菌株T42与Thermobrachium celere和Caloramator indicus位于同一分支,生理生化特征也表明菌株T42应是Thermobrachium属的一个新菌株,在中国普通微生物菌种保藏中心的保藏号为AS1.5039。菌株T42的最佳产氢初始pH为7.2,最佳产氢温度为62℃,其氢转化率为1.06mol H2/mol葡萄糖,最大产氢速率为24.0mmol H2/gDW/h。20mmol/L的Mg2 和2mmol/L的Fe2 可分别提高菌株T42的产氢量20%和23.3%,而Ni2 对其产氢无明显的作用。当菌株T42和热自养甲烷热杆菌(Methanothermobacter thermautotrophicus)Z245共培养时,由于降低了氢分压,使其葡萄糖利用率和氢产量分别提高1倍和2.8倍,发酵产物乙酸和乙醇的比例也从1提高到1.7。     

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。     

Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from d-glucose and α-d-glucose 1-phosphate (α-d-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to d-glucose and α-d-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35°C and 35 to 37.5°C, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only α-d-glucose 1-P served as a donor substrate with d-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the α-d-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with d-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5°C, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO₄ to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.Trehalose (1-α-d-glucopyranosyl-α-d-glucopyranoside) is a nonreducing disaccharide with an α,α-1,1 glycosidic linkage and is widely distributed in plants, insects, fungi, yeast, and bacteria (7). Due to the absence of reducing ends in trehalose, it is highly resistant to heat, pH, and Maillard’s reaction (24). In trehalose-producing organisms, this compound may serve as an energy reserve, a buffer against stresses such as desiccation and freezing, and a protein stabilizer (5, 7, 26, 31, 32). If trehalose can be produced economically, then it has potential commercial applications as a sweetener, a food stabilizer, and an additive in cosmetics and pharmaceuticals (6, 25). Recently, trehalose production through fermentation of yeast (17) and Corynebacterium (30), enzymatic processes from starch (18, 34) and maltose (19, 22, 23, 33), and extraction from transformed plants (10) has been reported.Our approach to trehalose production is to use an enzymatic process to produce trehalose from sucrose, one of the least expensive sugars. Since sucrose is efficiently converted to α-d-glucose 1-phosphate (α-d-glucose 1-P) and fructose by sucrose phosphorylase (SPase), we screened microorganisms for an enzyme that converts α-d-glucose 1-P to trehalose on the assumption that the combination of the putative trehalose synthase (TSase) and SPase would convert sucrose into trehalose. Although similar enzyme activities have been reported in the basidiomycete Flammulina velutipes (11) and in the yeast Pichia fermentans (27), these enzymes have not been well characterized.Our objectives were (i) to screen microorganisms, primarily fungi, for TSase activity; (ii) to purify and characterize the TSase; (iii) to identify the enzymatic process by which trehalose is produced from sucrose; and (iv) to identify an enzymatic process for production of trehalose from sucrose in which the fructose component is also converted to trehalose.     

Trehalose-6-P synthase is dispensable for growth on glucose but not for spore germination in Schizosaccharomyces pombe.

Trehalose-6-P inhibits hexokinases in Saccharomyces cerevisiae (M. A. Blázquez, R. Lagunas, C. Gancedo, and J. M. Gancedo, FEBS Lett. 329:51-54, 1993), and disruption of the TPS1 gene (formerly named CIF1 or FDP1) encoding trehalose-6-P synthase prevents growth in glucose. We have found that the hexokinase from Schizosaccharomyces pombe is not inhibited by trehalose-6-P even at a concentration of 3 mM. The highest internal concentration of trehalose-6-P that we measured in S. pombe was 0.75 mM after heat shock. We have isolated from S. pombe the tps1+ gene, which is homologous to the Saccharomyces cerevisiae TPS1 gene. The DNA sequence from tps1+ predicts a protein of 479 amino acids with 65% identity with the protein of S. cerevisiae. The tps1+ gene expressed from its own promoter could complement the lack of trehalose-6-P synthase in S. cerevisiae tps1 mutants. The TPS1 gene from S. cerevisiae could also restore trehalose synthesis in S. pombe tps1 mutants. A chromosomal disruption of the tps1+ gene in S. pombe did not have a noticeable effect on growth in glucose, in contrast with the disruption of TPS1 in S. cerevisiae. However, the disruption prevented germination of spores carrying it. The level of an RNA hybridizing with an internal probe of the tps1+ gene reached a maximum after 20 min of heat shock treatment. The results presented support the idea that trehalose-6-P plays a role in the control of glycolysis in S. cerevisiae but not in S. pombe and show that the trehalose pathway has different roles in the two yeast species.     

Role of trehalose in growth at high temperature of Salmonella enterica serovar Typhimurium

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of Salmonella enterica serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45 degrees C. One of these mutations proved to be in the seqA gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with pgm (which encodes phosphoglucomutase), it is likely to be polar on the expression of the pgm gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the seqA::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an otsA mutation, which inactivates the first step of the trehalose biosynthetic pathway. The otsA mutation caused a growth defect at 45 degrees C in minimal medium containing 0.2 M NaCl that was similar to that caused by the pgm mutation, but otsA did not affect growth rate in this medium at 37 degrees C. These results suggest that the growth defect of the seqA-pgm mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45 degrees C had a fivefold higher trehalose pool size than cells grown at 30 degrees C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.     

Trehalose phosphorylase from Pichia fermentans and its role in the metabolism of trehalose

During a screening for novel microbial trehalose phosphorylase three Pichia strains were identified as producers of this particular enzyme that have not yet been described. To our knowledge, this is the first time that this enzyme activity has been shown in yeasts. Pichia fermentans formed trehalose phosphorylase when cultivated on a growth medium containing easily metabolizable sugers such as glucose. Addition of NaCl (0.4 M) to the medium increased the synthesis of the enzyme significantly. Production of trehalose phosphorylase was found to be growth-associated with a maximum of activity formed at the transition of the exponential to the stationary phase of growth. Trehalose phosphorylase catalyzes the phosphorolytic cleavage of trehalose, yielding glucose 1-phosphate (glucose-1-P) and glucose as products. In vitro the enzyme readily catalyzes the reverse reaction, the synthesis of trehalose from glucose and glucose-1-P. For this reaction, the enzyme of P. fermentans was found to utilize -glucose-1-P preferentially. A partially purified enzyme preparation showed a pH optimum of 6.3 for the synthesis of trehalose. The enzyme was found to be rather unstable; it was easily inactivated by dilution unless Ca²⁺ or Mn²⁺ were added. This instability is presumably caused by dissociation of the enzyme. In contrast to other yeasts, P. fermentans rapidly degraded intracellularly accumulated trehalose when the carbon source in the medium was depleted. Trehalose phosphorylase seems to be a key enzyme in the degradative pathway of trehalose in P. fermentans. Additional enzymes in this catabolic pathway of trehalose include phosphoglucomutase, glucose-6-phosphate dehydrogenase, and gluconolactonase.This contribution is part of the Ph.D. thesis of Ingrid Schick     

The involvement of hexokinases in trehalose synthesis.

Yeast cells harboring a MAL2-8c gene accumulate trehalose during the transition phase of growth on glucose due to the presence of the ADPG-dependent trehalose 6-phosphate synthase. Under these conditions, glucokinase appeared not to provide G-6-P for trehalose synthesis and the two hexokinases seemed to act synergistically. After incubation in d-xylose, trehalose levels in these cells dropped almost in 90%, confirming the involvement of both hexokinases in the accumulation of this carbohydrate. Nevertheless, G-6-P levels appeared to be similar in all strains. Some explanations for this paradox are discussed. In stationary phase, neither of the three isoenzymes were involved in trehalose synthesis. Possibly, gluconeogenesis provides the substrate for trehalose synthesis at that stage.     

INS-1E细胞葡萄糖毒性模型的建立

目的:建立胰岛细胞系INS-1E细胞的葡萄糖毒性模型。方法:将INS-1E细胞分别在不同葡萄糖浓度(5.5 mmol/L、16.7mmol/L、25 mmol/L、30 mmol/L)的1640完全培养基中培养不同时间(48 h、72 h、96 h、120 h),分别在不同时间点取细胞进行细胞功能检测,实时荧光定量PCR法检测胰岛素m RNA的表达,ELISA检测葡萄糖刺激的胰岛素的分泌。结果:与对照组相比,高糖浓度(5.5 mmol/L、16.7 mmol/L、25 mmol/L、30 mmol/L)培养基中培养48 h后,INS-1E细胞的胰岛素合成和分泌的功能均增加(P均0.05),随着培养基中葡萄糖浓度的升高以及培养时间的延长,INS-1E细胞胰岛素合成及分泌的功能逐渐下降,当在葡萄糖浓度为30 mmol/L的培养基中培养120 h后,胰岛素m RNA合成及葡萄糖刺激的胰岛素分泌均显著降低(P均0.01)。结论:INS-1E细胞在30 m M的葡萄糖中培养120 h形成稳定的葡萄糖毒性模型。     

Pseudomonas putida S1海藻糖合成酶表达质粒的构建及诱导条件优化

采用PCR方法从Pseudomonas putida S1中克隆出编码海藻糖合成酶的基因treS,并与质粒pQE30T相连,构建了表达质粒pQE—TS2。将此重组质粒转化宿主菌E．coliM15进行诱导表达。十二烷基磺酸钠-聚丙烯酰胺凝胶SDS—PAGE电泳结果表明,treS基因在大肠杆菌中获得了高效表达。通过对诱导温度、诱导剂浓度、加诱导剂时间和诱导时间的优化研究,在菌液生长至OD600值为0．6时,加入诱导剂IPTG至终浓度0．01mmol／L,20℃诱导20h,蛋白的表达量达到每克干细胞89mg的蛋白,粗酶液酶活达到19U／mL。     

圆红冬孢酵母菌发酵产油脂培养基及发酵条件的优化研究

采用均匀设计和单因子试验法,系统考察了圆红冬孢酵母菌(Rhodosporidiumtoruloides)在不同碳氮比条件下产油发酵情况以及添加无机盐对产油发酵的影响,通过均匀设计软件对二次多项回归方程求解及单因素分析得知在培养基组成分别为葡萄糖70g/L,硫酸铵0.1g/L,酵母粉0.75g/L,磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,初始pH6.0,在灭菌(121℃15min)后添加ZnSO41.91×10-6mmol/L、CaCl21.50mmol/L、MnCl21.22×10-4mmol/L、CuSO41.00×10-4mmol/L。发酵摇瓶装液量为250mL三角瓶装培养基50mL,接种量为10%(种龄28h)。在上述条件下,30℃振荡(200r/min)培养120h,所得菌体油脂含量高达76.1%,脂肪得率系数可达22.7。     

培养基和培养条件对栀子悬浮细胞合成多糖的影响

对栀子悬浮细胞合成多糖的调控因子研究表明 :B5为最适培养基 ;5～10d继代周期的细胞可以保持良好的生长状态和多糖的合成能力 ;80g L的鲜细胞的接种量有利于栀子细胞的生长和多糖的合成 ;使用单一碳源时 ,葡萄糖比蔗糖对细胞生长更有益 ,但葡萄糖成本高 ,因而混合碳源 45g L(葡萄糖 :蔗糖 =1∶1)是最佳配方 ;氮源种类对细胞生长和多糖合成没有明显的影响 ,但氮源浓度是主要因素 ,40～50mmol L是最佳浓度 ,同时运用悬浮细胞生产栀子多糖可以通过在不同时间收获的细胞来避免提取时黄色素的干扰 ,具有很好的实际意义。     

腾冲嗜热厌氧菌热休克蛋白60及其基因表达的热激动态变化分析

热休克蛋白60(HSP60)是细菌体内一种非常重要的分子伴侣,其可以协助蛋白质或肽链的正确折叠和构型,防止变性和降解。基于本实验室的早期观察,腾冲嗜热厌氧菌的HSP60是一个典型的温度相关蛋白质,在80℃的表达水平最高。为了进一步了解嗜热菌应急的分子机制,继续进行了在热激后HSP60基因表达的动态研究。将最适温度(75℃)下培养的腾冲嗜热厌氧菌迅速地转移至80℃继续培养,然后在不同的时间点上分别取样,并通过双向电泳、Western blot和Real_time PCR等方法,分析了HSP60在mRNA和蛋白质水平上的表达量的改变。试验结果表明,在80℃热处理4h内的短期应急过程中,HSP60蛋白水平一直呈上升趋势,而它的mRNA水平则表现为先升高后下降的一个非对称性的峰形变化。HSP60的mRNA和蛋白质的对温度的应答快慢程度是不同的。HSP60的mRNA水平的显著变化在1h内便可观察到,而蛋白质水平的显著改变要延迟3h左右。此外,HSP60的mRNA和蛋白质对温度的应答量变大小也是不同的。     

Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.     

Enzymatic characterization of 5-methylthioribose 1-phosphate isomerase from Bacillus subtilis

The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P of 138 microM, and showed that the maximum velocity of the reaction was 20.4 micromol min(-1) (mg protein)(-1). The optimum reaction temperature and reaction pH were 35 degrees C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol(-1). The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.     

A kinetic analysis of glucokinase and glucose-6-P phosphatase in dictyostelium

Summary Using a mathematical model of carbohydrate metabolism in Dictyostelium discoideum, the kinetic expressions describing the activities of glucokinase and glucose-6-P phosphatase have been analyzed. The constraints on the kinetic mechanisms and relative activities of these two enzymes were investigated by comparing computer simulations to experimental data. The results indicated that, (1) glucose-6-P is compartmentalized with respect to the enzymes involved in glucose-6-P, trehalose and glycogen metabolism, (2) a differences of approximately 0.6 mm/min in maximum specific activity of glucokinase compared to glucose-6-P phosphatase is required in order for the model to produce end product carbohydrate levels consistent with those observed experimentally, (3) the Km of glucokinase for glucose strongly influences the steady state levels of glucose in the absence of external glucose, and (4) changing the order of product removal in the reaction catalyzed by glucose-6-P phosphatase influences the level of glycogen and trehalose.