Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.2.1.1 (ACS)
78,556 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acetate accumulation is a common problem observed in aerobic high cell density cultures of Escherichia coli. It has been hypothesized in previous reports that the glyoxylate shunt is active in E. coli BL21, the low acetate producer, and inactive in E. coli JM109, the high acetate producer. This hypothesis was further strengthened by incorporating 13C from uniformly labeled glucose into TCA cycle intermediates. Using northern blot analyses, the current report demonstrates that the reason for the inactivity of the glyoxylate pathway in E. coli JM109 is the no apparent transcription of isocitrate lyase (aceA) and malate synthase (aceB), and transcription of the isocitrate lyase repressor (iclR). The reverse is seen in E. coli BL21 where the glyoxylate pathway is active due to constitutive transcription of aceA and aceB and no transcription of the iclR. In addition, there is a difference between the two strains in the transcription of the acetyl-CoA synthetase (acs), phosphotransacetylase-acetate kinase (pta-ackA) pathway, and pyruvate oxidase (poxB), pathway. The transcript of acs is higher in E. coli BL21 and lower in the E. coli JM109, while the reverse is true for poxB transcription.
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PMID:Transcription levels of key metabolic genes are the cause for different glucose utilization pathways in E. coli B (BL21) and E. coli K (JM109). 1506 11

In a series of previous reports it was established by implementing metabolic flux, NMR/MS, and Northern blot analysis that the glyoxylate shunt, the TCA cycle, and acetate uptake by acetyl-CoA synthetase are more active in Escherichia coli BL21 than in Escherichia coli JM109. These differences were accepted as the reason for the differences in the glucose metabolism and acetate excretion of these two strains. Examination of the bacterial metabolism by microarrays and time course Northern blot showed that in addition to the glyoxylate shunt, the TCA cycle and the acetate uptake, other metabolic pathways are active differently in the two strains. These are gluconeogenesis, sfcA shunt, ppc shunt, glycogen biosynthesis, and fatty acid degradation. It was found that in E. coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses acetyl-CoA. The inactivation of the gluconeogenesis enzyme phosphoenolpyruvate synthetase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF) cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs, and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate concentration increased, but at low glucose concentrations these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis pathway (fbp, pckA, and ppsA) which leads to glycogen accumulation is constitutively active in E. coli BL21 regardless of glucose feeding strategy.
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PMID:Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses. 1580 47

The lipoamide dehydrogenase (LPD) encoded by lpdA gene is a component of the pyruvate dehydrogenase complex (PDHc), alpha-ketoglutarate dehydrogenase (AKGDH) and the glycine cleavage multi-enzyme (GCV) systems. In the present study, cell growth characteristics, enzyme activities and intracellular metabolite concentrations were compared between the parent strain Escherichia coli BW25113 and its lpdA knockout mutant in batch and continuous cultures. The lpdA knockout mutant produced significantly more pyruvate and L-glutamate under aerobiosis. Some D-lactate and succinate also accumulated in the culture broth. Based on the investigation of enzyme activities and intracellular metabolite concentrations, acetyl-CoA was considered to be formed by the combined reactions through pyruvate oxidase (PoxB), acetyl-CoA synthetase (Acs) and acetate kinase (Ack)-phosphoacetyltransferase (Pta) in the lpdA mutant. The effect of the lpdA gene knockout on the intracellular metabolic flux distributions was investigated based on 1H-13C NMR spectra and GC-MS signals obtained from 13C-labeling experiment using the mixture of [U-13C] glucose, [1-13C] glucose, and naturally labeled glucose. Flux analysis of the lpdA mutant indicated that the Entner-Doudoroff (ED) pathway and the glyoxylate shunt were activated. The fluxes through glycolysis and oxidative pentose phosphate (PP) pathway (except for the flux through glucose-6-phosphate dehydrogenase) were slightly downregulated. The TCA cycle was also downregulated in the mutant strain. On the other hand, the fluxes through the anaplerotic reactions of PEP carboxylase, PEP carboxykinase and malic enzyme were upregulated, which were consistent with the results of enzyme activities. Furthermore, the influence of the poxB gene knockout on the growth of E. coli was also studied because of its similar function to PDHc which connects the glycolysis to the TCA cycle. Under aerobiosis, a comparison of lpdA mutant and poxB mutant indicated that PDHc is the main enzyme which catalyzes the reaction from pyruvate to acetyl-CoA in the parent strain, while PoxB plays a very important role in the PDHc-deficient strain.
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PMID:Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments. 1631 Feb 73

The proteins of HL type cytoplasmic male sterility rice anther of YTA (CMS) and YTB (maintenance line) were separated by two-dimensional electrophoresis with immobilized ph (3-10 non-linear) gradients as the first dimension and SDS-PAGE as the second. The silver-stained proteins spots were analyzed using Image Master 2D software, there were about 1800 detectable spots on each 2D-gel, and about 85 spots were differential expressed. With direct MALDI-TOF mass spectrometry analysis and protein database searching, 9 protein spots out of 16 were identified. Among those proteins, there were Putative nucleic acid binding protein, glucose-1-phosphate adenylyltransferase (ADP-glucose pyrophosphorylase, AGPase) (EC: 2.7.7.27) large chain, UDP-glucuronic acid decarboxylase, putative calcium-binding protein annexin, putative acetyl-CoA synthetase and putative lipoamide dehydrogenase etc. They were closely associated with metabolism, protein biosynthesis, transcription, signal transduction and so on, all of which are cell activities that are essential to pollen development. Some of the identified proteins, i.e. AGPase, putative lipoamide dehydrogenase and putative acetyl-CoA synthetase were deeply discussed on the relationship to CMS. AGPase catalyzes a very important step in the biosynthesis of alpha 1,4-glucans (glycogen or starch) in bacteria and plants: synthesis of the activated glucosyl donor, ADP-glucose, from glucose-1-phosphate and ATP. The lack of the AGPase in male sterile line might directly result in the reduction of starch, and the synthesis of starch was the most important processes during the development of pollen. In present research, the descent or reduction of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase seemed involved in pollen sterility in rice. The degeneration and formation of various tissues during pollen development may impose high demands for energy and key biosynthetic intermediates. Under such conditions, the TCA cycle needs to operate fully, because the TCA cycle is an important source for many intermediates required for biosynthetic pathways, in addition to performing an oxidative, energy-producing role. Thus, it seemed reasonable to infer that the decrease of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase in anther might prevent the conversion of pyruvate into acetyl-CoA, and as a result, the TCA cycle could no longer operate at a sufficient rate to meet all requirements in anther cells, leading to pollen sterility. This study gave new insights into the mechanism of CMS in rice and demonstrated the power of the proteomic approach in plant biology studies.
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PMID:[Preliminary proteomics analysis of the total proteins of HL Type cytoplasmic male sterility rice anther]. 1655 98

This study investigated the subcellular localization of key enzymes of the glyoxylate cycle, i.e. isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (EC 2.3.3.9), that function constitutively in coordination with oxalate biosynthesis of glucose-grown Fomitopsis palustris. The ICL purified previously from F. palustris is termed FPICL1. Subcellular fractionation analysis of the cell homogenate by the sucrose density-gradient method showed that both key enzymes were present in peroxisomes, whereas acetyl-CoA synthase (EC 6.2.1.1) and oxalate-producing oxaloacetate acetylhydrolase (EC 3.7.1.1) were cytosolic. The peroxisomal localization of FPICL1 was further confirmed by electron microscopic and immunocytochemical analysis with anti-FPICL1 antibody. In addition, the peroxisomal target signal, composed of SKL at the C terminus of the cDNA encoding FPICL1, was found, which also suggests that FPICL1 is peroxisomal. Accordingly, it is postulated that transportation of succinate from peroxisomes to mitochondria, and vice versa, for the transportation of isocitrate or citrate, occurs in glucose-grown F. palustris for the constitutive metabolic coordination of the TCA and glyoxylate cycles with oxalate biosynthesis.
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PMID:Subcellular localization of glyoxylate cycle key enzymes involved in oxalate biosynthesis of wood-destroying basidiomycete Fomitopsis palustris grown on glucose. 1673 48

The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:plambdaFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.
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PMID:Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli. 1768 98

Mass production of glucosamine (GlcN) using microbial cells is a worthy approach to increase added values and keep safety problems in GlcN production process. Prior to set up a microbial cellular platform, this study was to assess acetate metabolism in Citrobacter sp. BL-4 (BL-4) which has produced a polyglucosamine PGB-2. The LC-MS analysis was conducted after protein separation on the 1D-PAGE to accomplish the purpose of this study. 280 proteins were totally identified and 188 proteins were separated as acetate-related proteins in BL-4. Acetate was converted to acetyl-CoA by acetyl-CoA synthetase up-regulated in the acetate medium. The glyoxylate bypass in the acetate medium was up-regulated with over-expression of isocitrate lyases and 2D-PAGE confirmed this differential expression. Using (1)H-NMR analysis, the product of isocitrate lyases, succinate, increased about 15 times in the acetate medium. During acetate metabolism proteins involved in the lipid metabolism and hexosamine biosynthesis were over-expressed in the acetate medium, while proteins involved in TCA cycle, pentose phosphate cycle and purine metabolism were down-regulated. Taken together, the results from the proteomic analysis can be applied to improve GlcN production and to develop metabolic engineering in BL-4.
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PMID:Proteomic analysis on acetate metabolism in Citrobacter sp. BL-4. 2221 Nov 6

Elucidating mechanisms of natural organofluorine biosynthesis is essential for a basic understanding of fluorine biochemistry in living systems as well as for expanding biological methods for fluorine incorporation into small molecules of interest. To meet this goal we have combined massively parallel sequencing technologies, genetic knockout, and in vitro biochemical approaches to investigate the fluoride response of the only known genetic host of an organofluorine-producing pathway, Streptomyces cattleya. Interestingly, we have discovered that the major mode of S. cattleya's resistance to the fluorinated toxin it produces, fluoroacetate, may be due to temporal control of production rather than the ability of the host's metabolic machinery to discriminate between fluorinated and non-fluorinated molecules. Indeed, neither the acetate kinase/phosphotransacetylase acetate assimilation pathway nor the TCA cycle enzymes (citrate synthase and aconitase) exclude fluorinated substrates based on in vitro biochemical characterization. Furthermore, disruption of the fluoroacetate resistance gene encoding a fluoroacetyl-CoA thioesterase (FlK) does not appear to lead to an observable growth defect related to organofluorine production. By showing that a switch in central metabolism can mediate and control molecular fluorine incorporation, our findings reveal a new potential strategy toward diversifying simple fluorinated building blocks into more complex products.
ACS Chem Biol 2012 Sep 21
PMID:Temporal and fluoride control of secondary metabolism regulates cellular organofluorine biosynthesis. 2276 62

We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs, encoding the acetyl coenzyme A synthetase, in Escherichia coli. We confirm most of the known genetic regulators, including CRP-cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA, sucB and lpdA, encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC. The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library.
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PMID:A genome-wide screen for identifying all regulators of a target gene. 2389 89

Parkinson's disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat "hijacks" the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression.
ACS Chem Biol 2014 Sep 19
PMID:Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity. 2493 2


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