Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is an important ubiquitous cytosol enzyme that fixes HCO3 together with phosphoenolpyruvate (PEP) and yields oxaloacetate that can be converted to intermediates of the citric acid cycle. In plant cells, PEPC participates in CO2 assimilation and other important metabolic pathways, and it has broad functions in different plant tissues. PEPC is also involved in the regulation of storage product synthesis and metabolism in seeds, such as affecting the metabolic fluxes from sugars/starch towards the synthesis of fatty acids or amino acids and proteins. In this review, we introduced the progress in classification, structure and regulation of PEPC in plant tissues. We discussed the potential applications of plant PEPCs in genetic engineering. The researches in functions and regulation mechanism of plant PEPCs will provide beneficial approaches to applications of plant PEPCs in high-yield crops breeding, energy crop and microbe genetic engineering.
Bicarbonates ; chemistry ; Genetic Engineering ; Oxaloacetic Acid ; chemistry ; Phosphoenolpyruvate ; chemistry ; Phosphoenolpyruvate Carboxylase ; chemistry ; genetics ; metabolism ; Plants ; enzymology

To realize the simultaneous fermentation of xylose and glucose, ptsG (one of the glucose-PTS genes) was deleted from the engineered ethanologenic Escherichia coli SZ470 (deltapflB, deltafrdABCD, deltaackA, deltaldhA), resulting in loss of glucose effect in the mutant SZ470P (deltaptsG). When tested in 5% mixture of glucose (2.5%) and xylose (2.5%), SZ470P simultaneously used glucose (13 g/L) and xylose (20 g/L) whereas the parent strain SZ470 sequentially used glucose (25 g/L) then xylose (5 g/L). Upon completion of the fermentation, both strains achieved similar product yield of 89%. SZ470P produced 15.01 g/L of ethanol, which was 14.32% higher than that produced by SZ470 (12.86 g/L). Deleting ptsG gene enabled the mutant strain SZ470P to simultaneously use both glucose and xylose and achieve better ethanol production.
Escherichia coli ; enzymology ; genetics ; Ethanol ; chemistry ; Fermentation ; Glucose ; chemistry ; Phosphoenolpyruvate Sugar Phosphotransferase System ; genetics ; Xylose ; chemistry

PURPOSE: Patients show variable responses to chemoradiotherapy (CRT), which is generally administered before surgery for locally advanced rectal cancer (LARC). The aim of this study was to identify molecular markers predictive of CRT responses by analysis of low-mass ions (LMIs) in serum of LARC patients. MATERIALS AND METHODS: LMIs (< 1,000 m/z) in serum obtained before CRT from 73 LARC (cT3-4) patients were profiled using matrix-assisted laser desorption/ionization mass spectrometry. LMIs with higher weighting factors in discriminating CRT responses were selected using principal components analysis and discriminant analysis. Selected LMIs were identified using the Human Metabolome Database. The concentrations of identified LMIs were determined by colorimetric enzyme assay, and compared according to post-CRT pathological stage (ypStage) or Dworak's tumor regression grade (TRG). RESULTS: The nine highest-ranking LMIs were selected. Among them, two LMIs with 137.08 and 169.04 m/z were identified as hypoxanthine (HX) and phosphoenolpyruvic acid (PEP), respectively. Higher HX concentration was observed in patients with ypStage 0-1 compared to ypStage 2-4 (p=0.034) or ypStage 3-4 (p=0.030); a similar difference was observed between TRG 4-3 and TRG 1 (p=0.035). HX > 16.0 muM showed significant association with ypStage 0-1 or TRG 4-3 than ypStage 3-4 (p=0.009) or TRG 1 (p=0.024), respectively. In contrast, a significantly lower concentration of PEP was observed in TRG 4-3 compared with TRG 2-1 (p=0.012). CONCLUSION: Findings of this study demonstrated that serum concentrations of HX and PEP, identified using LMI profiling, may be useful for predicting the CRT response of LARC patients before treatment.
Biological Markers* ; Chemoradiotherapy* ; Enzyme Assays ; Humans ; Hypoxanthine* ; Ions ; Mass Spectrometry ; Metabolome ; Phosphoenolpyruvate ; Rectal Neoplasms*

As an important enzyme for gluconeogenesis, mitochondrial phosphoenolpyruvate carboxykinase (PCK2) has further complex functions beyond regulation of glucose metabolism. Here, we report that conditional knockout of Pck2 in osteoblasts results in a pathological phenotype manifested as craniofacial malformation, long bone loss, and marrow adipocyte accumulation. Ablation of Pck2 alters the metabolic pathways of developing bone, particularly fatty acid metabolism. However, metformin treatment can mitigate skeletal dysplasia of embryonic and postnatal heterozygous knockout mice, at least partly via the AMPK signaling pathway. Collectively, these data illustrate that PCK2 is pivotal for bone development and metabolic homeostasis, and suggest that regulation of metformin-mediated signaling could provide a novel and practical strategy for treating metabolic skeletal dysfunction.
Mice ; Animals ; Metformin/pharmacology* ; Phosphoenolpyruvate Carboxykinase (ATP)/metabolism* ; Gluconeogenesis/genetics* ; Mice, Knockout

BACKGROUND/OBJECTIVES: Type 2 diabetes (T2D) is more frequently diagnosed and is characterized by hyperglycemia and insulin resistance. D-Xylose, a sucrase inhibitor, may be useful as a functional sugar complement to inhibit increases in blood glucose levels. The objective of this study was to investigate the anti-diabetic effects of D-xylose both in vitro and stretpozotocin (STZ)-nicotinamide (NA)-induced models in vivo. MATERIALS/METHODS: Wistar rats were divided into the following groups: (i) normal control; (ii) diabetic control; (iii) diabetic rats supplemented with a diet where 5% of the total sucrose content in the diet was replaced with D-xylose; and (iv) diabetic rats supplemented with a diet where 10% of the total sucrose content in the diet was replaced with D-xylose. These groups were maintained for two weeks. The effects of D-xylose on blood glucose levels were examined using oral glucose tolerance test, insulin secretion assays, histology of liver and pancreas tissues, and analysis of phosphoenolpyruvate carboxylase (PEPCK) expression in liver tissues of a STZ-NA-induced experimental rat model. Levels of glucose uptake and insulin secretion by differentiated C2C12 muscle cells and INS-1 pancreatic beta-cells were analyzed. RESULTS: In vivo, D-xylose supplementation significantly reduced fasting serum glucose levels (P < 0.05), it slightly reduced the area under the glucose curve, and increased insulin levels compared to the diabetic controls. D-Xylose supplementation enhanced the regeneration of pancreas tissue and improved the arrangement of hepatocytes compared to the diabetic controls. Lower levels of PEPCK were detected in the liver tissues of D-xylose-supplemented rats (P < 0.05). In vitro, both 2-NBDG uptake by C2C12 cells and insulin secretion by INS-1 cells were increased with D-xylose supplementation in a dose-dependent manner compared to treatment with glucose alone. CONCLUSIONS: In this study, D-xylose exerted anti-diabetic effects in vivo by regulating blood glucose levels via regeneration of damaged pancreas and liver tissues and regulation of PEPCK, a key rate-limiting enzyme in the process of gluconeogenesis. In vitro, D-xylose induced the uptake of glucose by muscle cells and the secretion of insulin cells by beta-cells. These mechanistic insights will facilitate the development of highly effective strategy for T2D.
Animals ; Blood Glucose* ; Complement System Proteins* ; Diet ; Fasting ; Gluconeogenesis ; Glucose Tolerance Test ; Glucose* ; Hepatocytes ; Hyperglycemia ; Insulin ; Insulin Resistance ; Liver ; Models, Animal ; Muscle Cells ; Pancreas ; Phosphoenolpyruvate Carboxylase* ; Phosphoenolpyruvate* ; Rats* ; Rats, Wistar ; Regeneration ; Sucrase ; Sucrose ; Xylose*

PURPOSE: Enolase is a cytoplasmic enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway. The aim of this study was to investigate whether the overexpression of neuron-specific enolase (NSE) can serve as a prognostic factor in patients with gastric cancer (GC). MATERIALS AND METHODS: To assess its prognostic value in GC, NSE expression was measured by immunohistochemistry in a clinically annotated tissue microarray comprising of 327 human GC specimens. Cytoplasmic NSE expression was scored from 0 to 4, reflecting the percentage of NSE-positive cells. RESULTS: In terms of histology as per the World Health Organization criteria (P=0.340), there were no differences between the NSE overexpression (NSE-OE) and NSE underexpression (NSE-UE) groups. The NSE-OE group showed a significantly lower rate of advanced GC (P<0.010), lymph node metastasis (P=0.010), advanced stage group (P<0.010), cancer-related death (P<0.010), and cancer recurrence (P<0.010). Additionally, a Kaplan-Meier survival analysis revealed that the NSE-OE group had longer cumulative survival times than the NSE-UE group (log-rank test, P<0.010). However, there were no significant differences in the serum levels of NSE expression in patients with GC and healthy volunteers (P=0.280). CONCLUSIONS: Patients with NSE overexpressing GC tissues showed better prognostic results, implying that NSE could be a candidate biomarker of GC.
Cytoplasm ; Healthy Volunteers ; Humans ; Immunohistochemistry ; Lymph Nodes ; Neoplasm Metastasis ; Phosphoenolpyruvate ; Phosphopyruvate Hydratase* ; Prognosis ; Recurrence ; Stomach Neoplasms* ; World Health Organization

Metabolic engineering provide powerful tools for the systematic manipulation of cellular metabolic activities. The ptsG gene for glucose-specific transporter Enzyme II CBGlc of the phosphotransferase system was knock-out so as to reduce the accumulation of acetic acid in the high cell-density culture of Escherichia coli on excess glucose. The chloramphenicol-resistant cassette with short shared sequences on both ends generated by PCR was electroporated into Escherichia coli DH5alpha and JM109. Recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombinase functions. Therefore, the ptsG gene was disrupted to construct the mutants called DH5alphaP and JM109P. There was no difference between the mutants and parent strains in LB media.However, in LB media supplemented with glucose, the mutants of Escherichia coli deficient in ptsG showed greater biomass, together with exploiting more glucose. The maximal cell density obtained with DH5alphaP was approximately 3 times more than that of DH5alpha, then the result of JM109P increased fourfold. The products of recombinant protein TNF respectively accounted for 24.3% of total cellular protein in DH5alphaP with A600 8.28 and 20.8% of total cellular protein in JM109P with A600 7.62. The specific volume expression amount of TNF was greater in the ptsG mutant than in its parent strain. These results demonstrate that the ptsG-mutant strains will be available for high cell-density culture.
Culture Media ; Escherichia coli ; genetics ; growth & development ; Fermentation ; Mutation ; Phosphoenolpyruvate Sugar Phosphotransferase System ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Tumor Necrosis Factor-alpha ; biosynthesis

In Bacillus subtilis , raising the amount of carbon catabolite in vivo would lead to carbon catabolite repression (CCR) and restrain the absorption of glucose. By deleting CcpA the CCR effect could be relieved, but the absorption of glucose remains restrained. The phosphoenol-pyruvate-sugar phosphotransferase system (PTS) is the main glucose transportation system in B. subtilis. HPr protein together with HprK/P participate in the glucose transportation. The HPr protein is phosphorylated at His-15 forming HPr-His-15-P transferring phosphate group from HPr to E II . While HprK/P phosphorylate HPr at Ser-46 forming HPr-Ser-46-P. HPr-Ser-46-P cannot participate in the transportation of glucose. The Knockout of ccpA gene increases the amount of fructose 1,6-bisphosphate(FBP) in vivo. And FBP could activate HPr kinase. So when CcpA is deleted, most part of the HPr will be phosphorylated at Ser-46. Absorpton of glucose is blocked. In this study, by disruption of hprk gene, the obtained B. subtilisZHc/pMX45 reaches the peak riboflavin production of 4.374mg/mL at the optimum glucose concentration of 10%, 19.2% higher than that of B. subtilis24 A1/pMX45 at the optimum glucose concentration of 8%.
Bacillus subtilis ; metabolism ; Bacterial Proteins ; genetics ; physiology ; Fermentation ; Glucose ; metabolism ; Mutation ; Phosphoenolpyruvate Sugar Phosphotransferase System ; genetics ; physiology ; Riboflavin ; metabolism ; Sucrose ; metabolism

Lactate and succinate were produced by Corynebacterium acetoacidophilum from glucose under oxygen deprivation conditions. To construct knockout mutant, lactate dehydrogenase gene (ldh) of C. acetoacidophilum was deleted by double-crossover chromosome replacement with sacB gene. Comparing with the wild strain ATCC13870, ldhA-deficent mutant produced no lactate with glucose consumption rate decreased by 29.3%, while succinate and acetate concentrations were increased by 45.6% and 182%, respectively. Moreover, the NADH/NAD+ rate was less than 1 (about 0.7), and the activities of phosphoenolpyruvate carboxylase and acetate kinase of the ldhA-deficent mutant were enhanced by 84% and 12 times, respectively. Our studies show that succinicate and acetate production pathways are strengthened by blocking lactate synthesis. It also suggests that improving NADH supply and eliminating acetate generation are alternative strategies to get high succinate-producer.
Corynebacterium glutamicum ; genetics ; metabolism ; Glucose ; Industrial Microbiology ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; metabolism ; Oxygen ; metabolism ; Phosphoenolpyruvate Carboxylase ; Succinic Acid ; metabolism

Salmonella causes a wide variety of diseases ranging from mild diarrhea to severe systemic infections, such as like typhoid fever, in multiple organisms, ranging from mice to humans. A lack of ptsI, which encodes the first component of phosphoenolpyruvate (PEP) : carbohydrate phosphotransferase system (PTS), is known to cause Salmonella Typhimurium attenuation; however, the mechanisms behind this have not yet been elucidated. In this study, a DNA microarray was performed to determine why the virulence of ptsI mutants is attenuated under low-oxygen conditions in which the ptsI expression is enhanced. Of 106 down-regulated genes, the most repressed were pdu and tdc genes, which are required for propanediol utilization and threonine and serine metabolism, respectively. In addition, half the flagellar genes were down-regulated in the ptsI mutant strain. Because pdu genes are induced during infection and Tdc products and flagella-mediated motility are necessary for the invasion of S. Typhimurium, the invasive ability of ptsI mutants was examined. We found that ptsI mutation reduced the ability of S. Typhimurium to invade into host cells, suggesting that reduced expression of the pdu, tdc, and flagellar genes is involved in the attenuation of ptsI mutants.
Animals ; Diarrhea ; Flagella ; Humans ; Metabolism ; Mice ; Microarray Analysis* ; Oligonucleotide Array Sequence Analysis ; Phosphoenolpyruvate ; Salmonella typhimurium* ; Salmonella* ; Serine ; Threonine ; Typhoid Fever ; Virulence