Involvement of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system in regulation of transcription of catabolic genes.
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Synthesis of catabolite-sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic AMP-receptor protein (crp mutation) Measurements of the differential rate of beta-galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains. It was concluded that mutational damage to the general components of the phosphoenolpyruvate-dependent phosphotransferase system diminishes activity of the lac promoter. The results obtained led to the conclusion that pts gene products (apparently phospho approximately HPr) are necessary for the initiation of transcription of catabolite-sensitive operons in E. coli. (+info)
In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis.
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Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates. Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3. (+info)
Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system.
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We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min(-1) mg of dry weight(-1). In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity. (+info)
Measurement of gluconeogenesis and mass isotopomer analysis based on [U-(13)C]glucose.
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Two methods of measuring rates of gluconeogenesis based on label redistribution after the introduction of [U-(13)C]glucose into the whole body are examined. These methods are compared with methods previously derived for carbon-14 tracers. It is shown that the three approaches (stoichiometric, dilution, and combinatorial) are equivalent, provided the same set of assumptions are used. Barring a factor of two [see Am. J. Physiol. 270 (Endocrinol. Metab. 33): E709-E717, 1996], the differences ( approximately 10-15%) in the carbon-based dilutional and the molecule-based estimates of the rate of gluconeogenesis from published isotopomer data likely arise from small differences in the assumptions that concern the relative rate of label loss from the different isotopomers. The production of unlabeled substrate for glucose synthesis (phosphoenolpyruvate) from the different isotopomers of lactate is shown to be a potential source of error in these methods. This error is estimated using models of the interaction of the gluconeogenetic pathway and the tricarboxylic acid (TCA) cycle and is shown to vary from negligible to 30% depending on the relative flux of the two pathways through the oxaloacetate pool. Because the estimates obtained by both methods considered are lower than is physiologically expected, some of the assumptions made may not hold. Future work will exploit the rich information content of isotopomer data to yield improved estimates. (+info)
Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli.
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We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP. (+info)
Estimating gluconeogenesis with [U-13C]glucose: molecular condensation requires a molecular approach.
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Recently three equations for estimating gluconeogenesis in vivo have been proposed, two by J. A. Tayek and J. Katz [Am. J. Physiol. 270 (Endocrinol. Metab. 33): E709-E717, 1996, and Am. J. Physiol. 272 (Endocrinol. Metab. 35): E476-E484, 1997] and one by B. R. Landau, J. Wahren, K. Ekberg, S. F. Previs, D. Yang, and H. Brunengraber [Am. J. Physiol. 274 (Endocrinol. Metab. 37): E954-E961, 1998]. Both groups estimate gluconeogenesis from cycling of [U-13C]glucose to lactate and back to glucose, detected by mass spectrometry. Landau's approach is based on analysis of labeled molecules, whereas Tayek and Katz's is based on labeling of carbon atoms by use of the concept of "molar enrichment," which weights each mass isotopomer by the number of labeled carbons. We derived an equation very similar to Landau's using binomial probability. Our analysis demonstrates that the molecular-based approach is correct. Additionally, equations appropriate for 14C studies are not appropriate for 13C studies, because the method used to detect 14C, decay of atoms, differs from 13C mass isotopomers detected as labeled molecules. We conclude that the molar enrichment carbon-based approach is not useful in the derivation of equations for the polymerization of molecules detected by mass spectrometry of molecules, and we confirm the findings of Landau et al. (+info)
The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.
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The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate. (+info)
Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations.
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The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(GLP versus PEP) up to 600-fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in bacterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram-positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad-spectrum antibacterial target. (+info)