Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of anaerobic glucose metabolism and lactate transport in Staphylococcus aureus cells

Ezra, F. S.; Lucas, Donald S.; Mustacich, Robert V.; Russell, Anne F.

doi:10.1021/bi00285a020

Cited by 44 publications

(33 citation statements)

References 39 publications

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“…As demonstrated above (Fig. 3 B), the two curves corresponding to ethanol production with and without the addition of unlabeled ethanol exhibit the same slope as can be expected from Eqns (1) and (4). Thus, ( F , -F2) = 0.40 mM/min.…”

Section: Determination Of F1 and F2 The Formation And Consumption Flsupporting

confidence: 79%

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by ¹H‐NMR and ¹³C‐NMR spectroscopy

Tran-Dinh

Hervé

Wietzerbin

1991

European Journal of Biochemistry

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We propose an experimental approach combining 'H-NMR and ' 3C-NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters: 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time. This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyl-CoA and glutamate via the Krebs cycle in glucose-grown repressed Saccharomyces cerevisiae cells fed with [1-' 3C]glucose and/or unlabeled ethanol. The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc. were studied qualitatively and/or quantitatively as a function of time. The 13C fractional enrichment of [2-'3C]ethanol was determined by observing the proton resonance of the methyl group. Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites. However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3. Apart from the fact that ethanol is a potential precursor of acetyl-CoA as expected, these results indicate that (a) the probability for citrate and 2-oxoglutarate to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent.The flux of ethanol formation from pyruvate is about three-times and nine-times greater than that of ethanol consumption and acetyl-CoA formation, respectively, from pyruvate via pyruvate dehydrogenase. Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2 + C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1 .I. By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption. The validity of the present model was supported by the data obtained from similar experiments using unlabeled ethanol and non-NMR techniques. Now that the investigation of cellular metabolism is in rapid progress, interest in understanding regulatory processes through measurement of the metabolite flux along the various pathways in cells is growing considerably. In recent years, thanks to the use of specifically labeled substrates, 13C-NMR was found to be a powerful tool for flux evaluation in isolated cells 11 -101, tissues [ll-151 and perfused organs [16-241 even though the I3C nucleus is much less sensitive than 'H and 31P. Many studies [I, 2, 9-11, 14, 16, 18-23] have been devoted to the flow of pyruvate and acetyl-CoA into the Krebs cycle frequently using the 13C fractional enrichment of some labeled compounds as experimental parameters. Chance et al.[I81 have developed a mathematical mode...

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Section: Determination Of F1 and F2 The Formation And Consumption Flsupporting

confidence: 79%

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by ¹H‐NMR and ¹³C‐NMR spectroscopy

Tran-Dinh

Hervé

Wietzerbin

1991

European Journal of Biochemistry

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“…Lactobacillus plantarum , 52 Deinococcus radiodurans , 182,183 B. subtilis , 62 and Staphylococcus aureus , 184 accumulate 20 mM, ~200 μM, ~100 μM, and >50 μM Mn II , respectively, under “normal” laboratory growth conditions. 185 In these organisms, the presence of high levels of Mn II would likely readily outcompete Fe II loading of a class Ia RNR, which may be reflected in the utilization of a class Ib RNR.…”

Section: Interplay Of Mn and Fe In Biological Systemsmentioning

confidence: 99%

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

2012

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How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use FeII as a Lewis acid under normal growth conditions but which switch to MnII under oxidative stress; (2) extradiol dioxygenases, which have been found to use both FeII and MnII, the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.

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“…Phosphorylation activity without phosphoenolpyruvate was 17 nmol´min 21 . P. multocida grown on mannitol exhibited mannitol 1-phosphate dehydrogenase activity of 840 nmol´min 21´m g 21 protein).…”

Section: Mannitol-pts Activity Mannitol 1-phosphate Dehydrogenase Anmentioning

confidence: 99%

“…For the 13 C NMR experiments, the pellets were suspended in K 2 HPO 4 /NaH 2 PO 4 to < 5 Â 10 10 cells´mL 21 . The cell suspension was then incubated in a NMR spectrometer at 30 8C.…”

Section: Preparation Of Whole Cells For 13 C Nmr Studiesmentioning

confidence: 99%

³¹P and ¹³C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida

Rager

Binet

Bouvet

1999

European Journal of Biochemistry

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Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13 C NMR spectroscopy, in cell-free extracts in vitro using 31 P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden±Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden±Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by a glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.

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Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of anaerobic glucose metabolism and lactate transport in Staphylococcus aureus cells

Cited by 44 publications

References 39 publications

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by ¹H‐NMR and ¹³C‐NMR spectroscopy

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by ¹H‐NMR and ¹³C‐NMR spectroscopy

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

³¹P and ¹³C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida

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Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of anaerobic glucose metabolism and lactate transport in Staphylococcus aureus cells

Cited by 44 publications

References 39 publications

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by 1H‐NMR and 13C‐NMR spectroscopy

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by 1H‐NMR and 13C‐NMR spectroscopy

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida

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Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by ¹H‐NMR and ¹³C‐NMR spectroscopy

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by ¹H‐NMR and ¹³C‐NMR spectroscopy

³¹P and ¹³C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida