Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae

We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant gamma-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including Delta(1)-pyrroline-5-carboxylate reductase, which converts Delta(1)-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant gamma-glutamyl kinase and the wild-type gamma-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Delta(1)-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.     

The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue

1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ;key' glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.     

巴氏杆菌糖酵解酶的黏附作用研究

巴氏杆菌（主要是多杀性巴氏杆菌）可以引起多种动物疫病（巴氏杆菌病）,同时也引起人类感染发病。[目的] 研究巴氏杆菌糖酵解酶对宿主细胞（兔肾细胞）和两种常见分子[纤连蛋白（fibronectin,Fn）和血浆纤维蛋白溶解酶原（plasminogen,Plg）]的黏附作用。[方法] 采用原核表达系统对多杀性巴氏杆菌的糖酵解酶进行表达并纯化及制备多克隆抗体,通过菌体表面蛋白定位检测、黏附与黏附抑制等实验探究巴氏杆菌糖酵解酶的黏附作用。[结果] 菌体表面蛋白检测结果显示除烯醇化酶和丙酮酸激酶外的7个糖酵解酶在多杀性巴氏杆菌表面存在。这7个糖酵解酶均能黏附兔肾细胞,但仅有磷酸葡萄糖异构酶的多克隆抗体能对多杀性巴氏杆菌黏附宿主细胞产生抑制作用。Far Western blotting结果显示9个糖酵解酶均能结合宿主Fn和Plg。招募抑制实验结果显示磷酸葡萄糖异构酶、醛缩酶、磷酸甘油酸变位酶的抗体对多杀性巴氏杆菌结合Fn和Plg都有抑制作用,磷酸果糖激酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶抗体仅对多杀性巴氏杆菌结合Fn或Plg有抑制作用。[结论] 多杀性巴氏杆菌糖酵解酶成员葡萄糖异构酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶、磷酸甘油酸变位酶在多杀性巴氏杆菌黏附宿主细胞或分子过程中发挥作用。该研究的完成将加深巴氏杆菌病分子发病机制的认识,并为巴氏杆菌病的诊断标识筛选、新型疫苗创制和药物靶标筛选等提供基础数据。     

Cloning metabolic pathway genes by complementation in Escherichia coli. Isolation and expression of Plasmodium falciparum glucose phosphate isomerase

Genetic complementation of an Escherichia coli double mutant was used to isolate and express the gene coding for Plasmodium falciparum glucose phosphate isomerase. The gene contains a 1773-base pair open reading frame, has no introns, and maps to P. falciparum chromosome 14. 34% of the deduced amino acid sequence is identical to human glucose phosphate isomerase, with highest similarity in regions of the proposed active sites. The putative initiation site of translation was determined by deletional and oligonucleotide mediated, site-specific mutageneses. Our data suggest that key metabolic enzymes of Plasmodia can be cloned and expressed in E. coli without prior knowledge of the primary amino acid or nucleic acid structure.     

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.     

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.     

Tracing Putative Trafficking of the Glycolytic Enzyme Enolase via SNARE-Driven Unconventional Secretion

Glycolytic enzymes are cytosolic proteins, but they also play important extracellular roles in cell-cell communication and infection. We used Saccharomyces cerevisiae to analyze the secretory pathway of some of these enzymes, including enolase, phosphoglucose isomerase, triose phosphate isomerase, and fructose 1,6-bisphosphate aldolase. Enolase, phosphoglucose isomerase, and an N-terminal 28-amino-acid-long fragment of enolase were secreted in a sec23-independent manner. The enhanced green fluorescent protein (EGFP)-conjugated enolase fragment formed cellular foci, some of which were found at the cell periphery. Therefore, we speculated that an overview of the secretory pathway could be gained by investigating the colocalization of the enolase fragment with intracellular proteins. The DsRed-conjugated enolase fragment colocalized with membrane proteins at the cis-Golgi complex, nucleus, endosome, and plasma membrane, but not the mitochondria. In addition, the secretion of full-length enolase was inhibited in a knockout mutant of the intracellular SNARE protein-coding gene TLG2. Our results suggest that enolase is secreted via a SNARE-dependent secretory pathway in S. cerevisiae.     

Regulation of fitness in yeast overexpressing glycolytic enzymes: responses to heat shock and nitrogen starvation.

Current models based on the analysis of linear metabolic pathways at steady-state predict that large increases over wild type in the activity of one enzyme will not alter an organism's fitness. This prediction is tested at steps in a highly branched pathway under two conditions known to alter steady-state: heat shock and nitrogen starvation. Saccharomyces cerevisiae transformants overproducing 1 of 4 enzymes in glycolysis (hexokinase B, phosphoglucose isomerase, phosphofructokinase, or pyruvate kinase) were subjected to heat shock in both exponential and stationary phases of growth. In neither phase does enzyme overexpression alter heat shock sensitivity. When starved for nitrogen in acetate medium, transformants overproducing hexokinase, phosphoglucose isomerase, and phosphofructokinase sporulate at the same rate and with the same frequency as cells harbouring only the plasmid vector. Current models therefore correctly predict the relationship between activity and components of fitness for 3 of 4 enzymes. By contrast, cells overexpressing pyruvate kinase sporulate poorly. This defect is not observed among cells transformed with a plasmid containing a Tn5 disrupted copy of the PYK gene. These findings are consistent with reports that implicate the PYK locus in yeast cell cycle control and suggest that it may be challenging to model relations between fitness and activity for multifunctional proteins.     

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.     

一个水稻双功能激酶基因的克隆及特征

从水稻中分离克隆出一个双功能激酶（OsSTY）基因,它编码一个含417个氨基酸的蛋白,其分子量为45926Da,等电点为7．689。OsSTY参与多种胁迫条件下的信号传导途径。低温或高温（4℃和37℃）处理后,OsSTY激酶的表达显著提高。机械损伤、水杨酸、乙烯等处理也促进该基因转录。另外,0sSTY基因在酿酒酵母（Saccharomyces cerevisiae）ste7基因缺失株（ste7／ste7）中的异源表达能够抑制该菌株在氮源饥饿条件下假菌丝生长的缺陷。Ste7是酿酒酵母的一个丝氨酸／苏氨酸／酪氨酸双功能激酶,它与OsSTY的激酶功能域有32％的同源性和50％的相似性。这揭示出OsSTY激酶能够校正,至少能部分地校正酿酒酵母ste7基因缺失株的假菌丝生长缺陷。     

Expression of E. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae

The Escherichia coli araBAD operon consists of three genes encoding three enzymes that convert L-arabinose to D-xylulose-5 phosphate. In this paper we report that the genes of the E. coli araBAD operon have been expressed in Saccharomyces cerevisiae using strong promoters from genes encoding S. cerevisiae glycolytic enzymes (pyruvate kinase, phosphoglucose isomerase, and phosphoglycerol kinase). The expression of these cloned genes in yeast was demonstrated by the presence of the active enzymes encoded by these cloned genes and by the presence of the corresponding mRNAs in the new host. The level of expression of L-ribulokinase (araB) and L-ribulose-5-phosphate 4-epimerase (araD) in S. cerevisiae was relatively high, with greater than 70% of the activity of the enzymes in wild type E. coli. On the other hand, the expression of L-arabinose isomerase (araA) reached only 10% of the activity of the same enzyme in wild type E. coli. Nevertheless, S. cerevisiae, bearing the cloned L-arabinose isomerase gene, converted L-arabinose to detectable levels of L-ribulose during fermentation. However, S. cerevisiae bearing all three genes (araA, araB, and araD) was not able to produce detectable amount of ethanol from L-arabinose. We speculate that factors such as pH, temperature, and competitive inhibition could reduce the activity of these enzymes to a lower level during fermentation compared to their activity measured in vitro. Thus, the ethanol produced from L-arabinose by recombinant yeast containing the expressed BAD genes is most likely totally consumed by the cell to maintain viability.     

Disruption of the fucose pathway as a consequence of genetic adaptation to propanediol as a carbon source in Escherichia coli.

In Escherichia coli, L-fucose is dissimilated via an inducible pathway mediated by L-fucose permease, L-fucose isomerase, L-fucose kinase, and L-fuculose 1-phosphate aldolase. The last enzyme cleaves the six-carbon substrate into dihydroxyacetone phosphate and L-lactaldehyde. Aerobically, lactaldehyde is oxidized to L-lactate by a nicotinamide adenine dinucleotide (NAD)-linked dehydrogenase. Anaerobically, lactaldehyde is reduced by an NADH-COUPLED REDUCTASE TO L-1,2-propanediol, which is lost into the medium irretrievably, even when oxygen is subsequently introduced. Propanediol excretion is thus the end result of a dismutation that permits further anaerobic metabolism of dihydroxy-acetone phosphate. A mutant selected for its ability to grow aerobically on propanediol as a carbon and energy source was reported to produce lactaldehyde reductase constitutively and at high levels, even aerobically. Under the new situation, this enzyme serves as a propanediol dehydrogenase. It was also reported that the mutant had lost the ability to grow on fucose. In the present study, it is shown that in wild-type cells the full synthesis of lactaldehyde dehydrogenase requires the presence of both molecular oxygen and a small molecule effector, and the full synthesis of lactaldehyde reductase requires anaerobiosis and the presence of a small molecule effector. The failure of mutant cells to grow on fucose reflects the impairment of a regulatory element in the fucose system that prevents the induction of the permease, the isomerase, and the kinase. The aldolase, on the other hand, is constitutively synthesized. Three independent fucose-utilizing revertants of the mutant all produce the permease, the isomerase, the kinase, as well as the aldolase, constitutively. These strains grow less well than the parental mutant on propanediol.     

Diacylglycerol kinase defect in a Drosophila retinal degeneration mutant rdgA

The Drosophila visual mutant rdgA is known to show age-dependent retinal degeneration with defective diacylglycerol (DG) kinase activity. In this study we examined DG kinase activity of several visual mutants and found that only rdgA mutant eyes showed the lack of DG kinase activity in a gene dosage-dependent manner. The enzyme activity is already absent at the time of eclosion from pupal case when the degeneration is not yet apparent. To examine whether rdgA gene dosage effect holds for other enzymes related to the phosphatidylinositol turnover, phospholipase C was analyzed which did not show any gene dosage effect. Therefore, it is strongly suggested that rdgA gene correlates closely with DG kinase activity, and the defect of DG kinase activity is a primary cause of retinal degeneration in rdgA mutant.     

Crystal structure of muconate lactonizing enzyme at 3 A resolution

The crystal structure of muconate lactonizing enzyme has been solved at 3 A resolution, and an unambiguous alpha-carbon backbone chain trace made. The enzyme contains three domains; the central domain is a parallel-stranded alpha-beta barrel, which has previously been reported in six other enzymes, including triose phosphate isomerase and pyruvate kinase. One novel feature of this enzyme is that its alpha-beta barrel has only seven parallel alpha-helices around the central core of eight parallel beta-strands; all other known alpha-beta barrels contain eight such helices. The N-terminal (alpha + beta) and C-terminal domains cover the cleft where the eighth helix would be. The active site of muconate lactonizing enzyme has been found by locating the manganese ion that is essential for catalytic activity, and by binding and locating an inhibitor, alpha-ketoglutarate. The active site lies in a cleft between the N-terminal and barrel domains; when the active sites of muconate lactonizing enzyme and triose phosphate isomerase are superimposed, barrel-strand 1 of triose phosphate isomerase is aligned with barrel-strand 3 of muconate lactonizing enzyme. This implies that structurally homologous active-site residues in the two enzymes are carried on different parts of the primary sequence; the ancestral gene would had to have been transposed during its evolution to the modern proteins, which seems unlikely. Therefore, these two enzymes may be related by convergent, rather than divergent, evolution.     

Identification of Mycoplasma pirum genes involved in the salvage pathways for nucleosides.

Genes encoding enzymes involved in the salvage pathway for nucleosides have been cloned and sequenced from the mollicute Mycoplasma pirum. One of them, encoding deoxyriboaldolase, was functionally identified by complementation of an Escherichia coli mutant. These genes are clustered, suggesting an operon organization, and they are immediately followed by the putative gene for the triose phosphate isomerase, an enzyme used during glycolysis.     

Characterization of mutants with single and multiple defects in the tryptophan biosynthetic pathway in Bacillus subtilis

Sixty-five tryptophan auxotrophs which map in a cluster on the genome of Bacillus subtilis were characterized on the basis of (i) growth response, (ii) accumulation of intermediate compounds, and (iii) determination of enzymatic defects. They could be placed into six phenotypic classes. Certain of the mutants exhibited pleiotropic effects on more than one enzymatic activity in a manner different from those effects reported for the tryptophan pathway in other organisms. Invariably, mutations in the second gene, that coding for phosphoribosyl transferase activity, were found to lack the indoleglycerol phosphate synthase activity specified by the third gene in the cluster; however, this polarity did not extend to genes more distal in the cluster. Furthermore, mutations in the gene which codes for phosphoribosyl-anthranilate isomerase not only led to a loss of this enzyme but also to a loss of phosphoribosyl transferase and indoleglycerol phosphate synthase. In contrast, mutations in either of the loci coding for these latter functions had no apparent effect on isomerase activity. No polarity of the conventional type was found, e.g., none of the mutations in any gene led to polarized effects on the levels of the enzymes specified by the other genes of the cluster. These observations indicated a possible in vivo aggregation involving the transferase, isomerase, and synthase enzymes, with the isomerase acting as the "key" enzyme in the aggregate.     

Enzyme polymorphism in Mansonia uniformis, a mosquito vector of brugian filariasis

Three temporal samples of a wild population of Mansonia uniformis were analysed for genetic variation at six gene-enzyme systems. Adenylate kinase, hexokinase (3 loci) and cathodal malate dehydrogenase were monomorphic. Phosphoglucomutase, glucose phosphate isomerase, isocitrate dehydrogenase and anodal malate dehydrogenase were polymorphic. Each of the polymorphic loci was represented by three alleles. The average heterozygosity or gene diversity was 0.0437.     

Development of a selection system for the detection of L-ribose isomerase expressing mutants of <Emphasis Type="Italic">Escherichia coli</Emphasis>

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.