Ultrahigh Performance Liquid Chromatography−Tandem Mass Spectrometry Method for Fast and Robust Quantification of Anionic and Aromatic Metabolites

Buescher, Joerg M.; Moco, Sofia; Sauer, Uwe; Zamboni, Nicola

doi:10.1021/ac100101d

Cited by 328 publications

(306 citation statements)

References 36 publications

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“…1B). Next, we determined the FBP concentrations at the different glycolytic fluxes using a targeted metabolomics approach (17) and found a positive, linear correlation of this metabolite's concentration with the glycolytic flux over a wide range of flux values (Fig. 1C), in agreement with what was observed earlier (18).…”

Section: Resultssupporting

confidence: 84%

Functioning of a metabolic flux sensor in Escherichia coli

Kochanowski

Volkmer

Gerosa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

191

183

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Regulation of metabolic operation in response to extracellular cues is crucial for cells' survival. Next to the canonical nutrient sensors, which measure the concentration of nutrients, recently intracellular "metabolic flux" was proposed as a novel impetus for metabolic regulation. According to this concept, cells would have molecular systems ("flux sensors") in place that regulate metabolism as a function of the actually occurring metabolic fluxes. Although this resembles an appealing concept, we have not had any experimental evidence for the existence of flux sensors and also we have not known how these flux sensors would work in detail. Here, we show experimental evidence that supports the hypothesis that Escherichia coli is indeed able to measure its glycolytic flux and uses this signal for metabolic regulation. Combining experiment and theory, we show how this flux-sensing function could emerge from an aggregate of several molecular mechanisms: First, the system of reactions of lower glycolysis and the feedforward activation of fructose-1,6-bisphosphate on pyruvate kinase translate flux information into the concentration of the metabolite fructose-1,6-bisphosphate. The interaction of this "flux-signaling metabolite" with the transcription factor Cra then leads to flux-dependent regulation. By responding to glycolytic flux, rather than to the concentration of individual carbon sources, the cell may minimize sensing and regulatory expenses. R egulation of metabolic operation is crucial for cells' survival. The canonical view is that this regulation occurs in response to extracellular cues, where, for instance, nutrient-specific transmembrane or intracellular receptors sense the presence of a nutrient and transfer respective commands to the regulatory machinery (1-5).Recently, however, a novel impetus for metabolic regulation was proposed: cells could regulate their metabolism as a function of the actually occurring intracellular metabolic fluxes (6, 7). According to this concept, changes in extracellular nutrient abundances would first-in a rather passive manner-result in changes in intracellular metabolic fluxes. In a second instance, the metabolic fluxes would be sensed by molecular systems ("flux sensors"), which in turn would transmit the sensed "flux signal" to the regulatory machinery that consequently would adjust metabolic operation (6). On the basis of a detailed mathematical model of Escherichia coli's central metabolism and its regulation, Kotte et al. suggested that this organism would have such flux sensors in place that establish a correlation between a metabolic flux and the concentration of certain so-called flux-signaling metabolites, which in turn affect the activity of transcription factors and thus would allow for transcriptional regulation in a flux-dependent manner.Using intracellular flux to regulate metabolism is an appealing concept as it would omit the need for nutrient-specific sensors for many different nutrients, and would allow the integration of multiple nutrient inputs directl...

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Section: Resultssupporting

confidence: 84%

Functioning of a metabolic flux sensor in Escherichia coli

Kochanowski

Volkmer

Gerosa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

191

183

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“…The fast centrifugation method from Kleijn et al was adapted for use with cell extracts (35). LC-MS/MS analysis was performed as in Buescher et al with minor adjustments (34). Internal standard curves made with 12 C compounds were used for quantification.…”

Section: Enzyme Engineeringmentioning

confidence: 99%

Computational protein design enables a novel one-carbon assimilation pathway

Siegel¹,

Smith²,

Poust

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

323

255

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We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.computational protein design | pathway engineering | carbon fixation N ovel strategies are needed to address current challenges in energy storage and carbon sequestration. One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1-3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).An optimal pathway for one-carbon utilization in common synthetic biology platforms would be (i) composed of a minimal number of enzymes, (ii) linear and disconnected from other metabolic pathways, (iii) thermodynamically favorable with a significant driving force at most or all steps, and (iv) capable of functioning in a robust manner under both aerobic and anaerobic conditions (5). A pathway with these properties could enable the assimilation of one-carbon molecules as the sole carbon source for the production of fuels and chemicals. Although no such pathway is known in nature, the established electrochemical reduction of carbon dioxide to formate under ambient temperatures and pressures in neutral aqueous solutions provides an attractive starting point for a onecarbon fixation pathway (5-8).We describe the computational design of an enzyme that catalyzes the carboligation of three one-carbon molecules into a single three-carbon molecule. This enzyme enables the construction of a new pathway, the formolase pathway, in which formate is converted into the central metabolite dihydroxyacetone phosphate (DHAP; Fig. 1). The use of computational protein design to reengineer catalytic activities opens up the pathway design space beyond that available based o...

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“…Internal standards (norvaline and glutaric acid) were added before drying. Intracellular metabolites were analyzed by ion-pairing ultrahighperformance liquid chromatography-tandem mass spectrometry (43). Extracellular metabolites were analyzed by gas chromatography-time of flight-mass spectrometry after off-line methoxyamination and just-in-time N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide derivatization.…”

Section: Metabolite Extraction and Analysis By Mass Spectrometrymentioning

confidence: 99%

Functional Metabolic Screen Identifies 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 as an Important Regulator of Prostate Cancer Cell Survival

Ros

Santos²,

Moco³

et al. 2012

Cancer Discovery

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178

182

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In this study we used an unbiased functional approach to identify metabolic enzymes required for the survival of prostate cancer cells. Expression of glycolytic and lipogenic enzymes is induced in response to androgens in prostate cancer (12). Prostate cancers also show a high rate of de novo lipogenesis, and inhibition of this process decreases the viability of prostate cancer cells (13,14). The prostate epithelium secretes large amounts of citrate into the seminal fluid, which is achieved by inhibition of aconitase, resulting in a truncated tricarboxylic acid cycle. Interestingly, during transformation, aconitase is reactivated, and prostate cancer cells oxidize citrate to generate energy (15). Because many cancer cells exhibit attenuated mitochondrial oxidative metabolism (16), prostate cancer may represent a unique metabolic situation.The characterization of several metabolic features of nonmalignant and prostate cancer cell lines presented here shows that the latter are glucose dependent when cultured both in full medium (FM) or in lipid-depleted medium (LDM) and sensitive to lipid synthesis inhibition when cultured in LDM. Using these cells in a siRNA screen targeting basic glucose metabolism, lipogenesis, and amino acid biosynthesis genes, in both full medium (FM) and in LDM, we were able to identify several genes selectively required for the survival of the prostate cancer cell lines. Among these was 6-phosphofructo-2-kinase/ fructose-2,6-biphosphatase 4 (PFKFB4), an isoform of the glycolytic enzyme phosphofructokinase 2 (PFK2). A detailed analysis of the role of this gene in prostate cancer cell survival revealed that PFKFB4 is required to maintain redox balance and to support tumor growth in vivo, highlighting the importance of the metabolic regulation of bioenergetics and antioxidant production in cancer cells. RESULTS Metabolic Characterization of Prostate Cell LinesTo identify metabolic weaknesses of prostate cancer cells, we first characterized the metabolic requirements of 2 Alterations in metabolic activity contribute to the proliferation and survival of cancer cells. We investigated the effect of siRNA-mediated gene silencing of 222 metabolic enzymes, transporters, and regulators on the survival of 3 metastatic prostate cancer cell lines and a nonmalignant prostate epithelial cell line. This approach revealed significant complexity in the metabolic requirements of prostate cancer cells and identified several genes selectively required for their survival. Among these genes was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), an isoform of phosphofructokinase 2 (PFK2). We show that PFKFB4 is required to balance glycolytic activity and antioxidant production to maintain cellular redox balance in prostate cancer cells. Depletion of PFKFB4 inhibited tumor growth in a xenograft model, indicating that it is required under physiologic nutrient levels. PFKFB4 mRNA expression was also found to be greater in metastatic prostate cancer compared with primary tumors. Taken together, these resul...

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Ultrahigh Performance Liquid Chromatography−Tandem Mass Spectrometry Method for Fast and Robust Quantification of Anionic and Aromatic Metabolites

Cited by 328 publications

References 36 publications

Functioning of a metabolic flux sensor in Escherichia coli

Functioning of a metabolic flux sensor in Escherichia coli

Computational protein design enables a novel one-carbon assimilation pathway

Functional Metabolic Screen Identifies 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 as an Important Regulator of Prostate Cancer Cell Survival

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