Kinetic modelling of glycolytic oscillations.

Glycolytic oscillations have been studied for well over 60 years, but aspects of their function, and mechanisms of regulation and synchronisation remain unclear. Glycolysis is amenable to mechanistic mathematical modelling, as its components have been well characterised, and the system can be studied at many organisational levels: in vitro reconstituted enzymes, cell free extracts, individual cells, and cell populations. In recent years, the emergence of individual cell analysis has opened new ways of studying this intriguing system.

Inhibitor titrations reveal low control of glyceraldehyde 3-phosphate dehydrogenase and high control of hexokinase on glycolytic flux in an aggressive triple-negative breast cancer cell line.

The glycolytic flux, and in particular lactate production, is strongly increased in cancer cells compared to normal cells, a characteristic often referred to as aerobic glycolysis or the Warburg effect. This makes the glycolytic pathway a potential drug target, in particular if the flux control distribution in the pathway has shifted due to the metabolic reprogramming in cancer cells. The flux response of a drug is dependent on both the sensitivity of the target to the drug and the flux control of the target, and both these characteristics can be exploited to obtain selectivity for cancer cells. Traditionally drug development programs have focused on selective sensitivity of the drug, not necessarily focussing on the flux control of the target. We determined the flux control of two steps that have been suggested to have high control in cancer cells, using two inhibitors, iodoacetic acid and 3-bromopyruvate, and measured a flux control of the glyceraldehyde 3-phosphate dehydrogenase close to zero, while the hexokinase holds 50% of all flux control in glycolysis in an invasive cancer cell line MDA-mb-231.

N-Myc-induced metabolic rewiring creates novel therapeutic vulnerabilities in neuroblastoma.

N-Myc is a transcription factor that is aberrantly expressed in many tumor types and is often correlated with poor patient prognosis. Recently, several lines of evidence pointed to the fact that oncogenic activation of Myc family proteins is concomitant with reprogramming of tumor cells to cope with an enhanced need for metabolites during cell growth. These adaptions are driven by the ability of Myc proteins to act as transcriptional amplifiers in a tissue-of-origin specific manner. Here, we describe the effects of N-Myc overexpression on metabolic reprogramming in neuroblastoma cells. Ectopic expression of N-Myc induced a glycolytic switch that was concomitant with enhanced sensitivity towards 2-deoxyglucose, an inhibitor of glycolysis. Moreover, global metabolic profiling revealed extensive alterations in the cellular metabolome resulting from overexpression of N-Myc. Limited supply with either of the two main carbon sources, glucose or glutamine, resulted in distinct shifts in steady-state metabolite levels and significant changes in glutathione metabolism. Interestingly, interference with glutamine-glutamate conversion preferentially blocked proliferation of N-Myc overexpressing cells, when glutamine levels were reduced. Thus, our study uncovered N-Myc induction and nutrient levels as important metabolic master switches in neuroblastoma cells and identified critical nodes that restrict tumor cell proliferation.

The Carbon Switch at the Level of Pyruvate and Phosphoenolpyruvate in Sulfolobus solfataricus P2.

Sulfolobus solfataricus P2 grows on different carbohydrates as well as alcohols, peptides and amino acids. Carbohydrates such as D-glucose or D-galactose are degraded via the modified, branched Entner-Doudoroff (ED) pathway whereas growth on peptides requires the Embden-Meyerhof-Parnas (EMP) pathway for gluconeogenesis. As for most hyperthermophilic Archaea an important control point is established at the level of triosephophate conversion, however, the regulation at the level of pyruvate/phosphoenolpyruvate conversion was not tackled so far. Here we describe the cloning, expression, purification and characterization of the pyruvate kinase (PK, SSO0981) and the phosphoenolpyruvate synthetase (PEPS, SSO0883) of Sul. solfataricus. The PK showed only catabolic activity [catalytic efficiency (PEP): 627.95 mM-1s-1, 70°C] with phosphoenolpyruvate as substrate and ADP as phosphate acceptor and was allosterically inhibited by ATP and isocitrate (K i 0.8 mM). The PEPS was reversible, however, exhibited preferred activity in the gluconeogenic direction [catalytic efficiency (pyruvate): 1.04 mM-1s-1, 70°C] and showed some inhibition by AMP and α-ketoglutarate. The gene SSO2829 annotated as PEPS/pyruvate:phosphate dikinase (PPDK) revealed neither PEPS nor PPDK activity. Our studies suggest that the energy charge of the cell as well as the availability of building blocks in the citric acid cycle and the carbon/nitrogen balance plays a major role in the Sul. solfataricus carbon switch. The comparison of regulatory features of well-studied hyperthermophilic Archaea reveals a close link and sophisticated coordination between the respective sugar kinases and the kinetic and regulatory properties of the enzymes at the level of PEP-pyruvate conversion.

Phosphoglycerate kinase acts as a futile cycle at high temperature.

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

Systems biology of the modified branched Entner-Doudoroff pathway in Sulfolobus solfataricus.

Sulfolobus solfataricus is a thermoacidophilic Archaeon that thrives in terrestrial hot springs (solfatares) with optimal growth at 80°C and pH 2-4. It catabolizes specific carbon sources, such as D-glucose, to pyruvate via the modified Entner-Doudoroff (ED) pathway. This pathway has two parallel branches, the semi-phosphorylative and the non-phosphorylative. However, the strategy of S.solfataricus to endure in such an extreme environment in terms of robustness and adaptation is not yet completely understood. Here, we present the first dynamic mathematical model of the ED pathway parameterized with quantitative experimental data. These data consist of enzyme activities of the branched pathway at 70°C and 80°C and of metabolomics data at the same temperatures for the wild type and for a metabolic engineered knockout of the semi-phosphorylative branch. We use the validated model to address two questions: 1. Is this system more robust to perturbations at its optimal growth temperature? 2. Is the ED robust to deletion and perturbations? We employed a systems biology approach to answer these questions and to gain further knowledge on the emergent properties of this biological system. Specifically, we applied deterministic and stochastic approaches to study the sensitivity and robustness of the system, respectively. The mathematical model we present here, shows that: 1. Steady state metabolite concentrations of the ED pathway are consistently more robust to stochastic internal perturbations at 80°C than at 70°C; 2. These metabolite concentrations are highly robust when faced with the knockout of either branch. Connected with this observation, these two branches show different properties at the level of metabolite production and flux control. These new results reveal how enzyme kinetics and metabolomics synergizes with mathematical modelling to unveil new systemic properties of the ED pathway in S.solfataricus in terms of its adaptation and robustness.

The Peculiar Glycolytic Pathway in Hyperthermophylic Archaea: Understanding Its Whims by Experimentation In Silico.

Mathematical models are key to systems biology where they typically describe the topology and dynamics of biological networks, listing biochemical entities and their relationships with one another. Some (hyper)thermophilic Archaea contain an enzyme, called non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), which catalyzes the direct oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate omitting adenosine 5'-triphosphate (ATP) formation by substrate-level-phosphorylation via phosphoglycerate kinase. In this study we formulate three hypotheses that could explain functionally why GAPN exists in these Archaea, and then construct and use mathematical models to test these three hypotheses. We used kinetic parameters of enzymes of Sulfolobus solfataricus (S. solfataricus) which is a thermo-acidophilic archaeon that grows optimally between 60 and 90 °C and between pH 2 and 4. For comparison, we used a model of Saccharomyces cerevisiae (S. cerevisiae), an organism that can live at moderate temperatures. We find that both the first hypothesis, i.e., that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plus phosphoglycerate kinase (PGK) route (the alternative to GAPN) is thermodynamically too much uphill and the third hypothesis, i.e., that GAPDH plus PGK are required to carry the flux in the gluconeogenic direction, are correct. The second hypothesis, i.e., that the GAPDH plus PGK route delivers less than the 1 ATP per pyruvate that is delivered by the GAPN route, is only correct when GAPDH reaction has a high rate and 1,3-bis-phosphoglycerate (BPG) spontaneously degrades to 3PG at a high rate.

A systems biology approach reveals major metabolic changes in the thermoacidophilic archaeon Sulfolobus solfataricus in response to the carbon source L-fucose versus D-glucose.

Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, the growth on different carbon sources was analysed, using an integrated systems biology approach. The comparison of growth on L-fucose and D-glucose allows first insights into the genome-wide changes in response to the two carbon sources and revealed a new pathway for L-fucose degradation in S. solfataricus. During growth on L-fucose major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3-hydroxypropionate/4-hydroxybutyrate cycle were observed. Within the newly discovered pathway for L-fucose degradation the following key reactions were identified: (i) L-fucose oxidation to L-fuconate via a dehydrogenase, (ii) dehydration to 2-keto-3-deoxy-L-fuconate via dehydratase, (iii) 2-keto-3-deoxy-L-fuconate cleavage to pyruvate and L-lactaldehyde via aldolase and (iv) L-lactaldehyde conversion to L-lactate via aldehyde dehydrogenase. This pathway as well as L-fucose transport shows interesting overlaps to the D-arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species.

One-step synthesis of 2-keto-3-deoxy-d-gluconate by biocatalytic dehydration of d-gluconate.

2-Keto-3-deoxy-sugar acids are key intermediates of central metabolism and integral constituents of bacterial (lipo)polysaccharides and cell wall components and are therefore continuously and highly demanded in related research fields. The stereospecific chemical synthesis of chiral 2-keto-deoxy-sugar acids involves a multitude of reaction steps, while in metabolic pathways only few conversions lead to the same 2-keto-3-deoxy sugar acids from easily available carbohydrate precursors. Here we present a straightforward and highly economic one-step biocatalytic synthesis procedure of 2-keto-3-deoxy-d-gluconate (KDG) from d-gluconate using recombinant gluconate dehydratase (GAD) from the hyperthermophilic crenarchaeon Thermoproteus tenax. This method is highly advantageous to KDG production schemes described so far for several reasons: (i) the d-gluconate is completely converted to stereochemically pure D-KDG without side-product formation, (ii) the final KDG yield is approximately 90%, (iii) the newly developed quantitative and qualitative LC-MS analysis method enabled the simultaneous detection of d-gluconate and KDG and (iv) the T. tenax GAD as biocatalyst can be provided by a simple and rapid procedure involving only two precipitation steps. The described utilization of dehydratases for 2-keto-3-deoxy sugar acid syntheses represents a highly resource-efficient one-step preparation and offers potential short synthetic routes toward a broad range of 2-keto-3-deoxy sugar acids and their derivatives.

Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus.

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/phosphatase, maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1-h period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the system fluxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency.

Unraveling the function of the two Entner-Doudoroff branches in the thermoacidophilic Crenarchaeon Sulfolobus solfataricus P2.

Sulfolobus solfataricus P2 is a thermoacidophilic archaeon that metabolizes glucose and galactose via an unusual branched Entner-Doudoroff (ED) pathway, which is characterized by a non-phosphorylative (np) and a semi-phosphorylative (sp) branch. However, so far the physiological significance of the two pathway branches is unknown. In order to address these questions two key enzymes of the branched ED pathway, the class II glycerate kinase (GK) of the np-ED branch and the 2-keto-3-deoxygluconate kinase (KDGK) of the sp-ED branch in S. solfataricus, were investigated. GK was recombinantly purified and characterized with respect to its kinetic properties. Mg(2+) dependent Sso-GK (glycerate + ATP → 2-phosphoglycerate + ADP) showed unusual regulatory properties, i.e. substrate inhibition and cooperativity by D-glycerate and ATP, and a substrate-inhibition model was established fitting closely to the experimental data. Furthermore, deletion of the sp-ED key enzyme KDGK in S. solfataricus PBL2025 resulted in a similar growth phenotype on glucose as substrate compared with the wild-type. In contrast, the mutant showed strongly increased concentrations of np-ED intermediates whereas the hexose and pentose phosphates as well as trehalose were decreased. Together the results indicate (a) that the np-ED pathway is able to compensate for the missing sp-ED branch in glucose catabolism, (b) that in addition to its catabolic function the sp-ED pathway has an additional although not essential role in providing sugar phosphates for anabolism/gluconeogenesis and (c) that GK, with its unusual regulatory properties, seems to play a major role in controlling the flux between the glycolytic np-ED and the glycolytic/gluconeogenetic sp-ED pathway.

Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences.

The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation using the arCOGs database, plus other available functional, structural and phylogenetic information. The goal of this initiative is continuous improvement of genome annotation with the support of the Sulfolobus research community.

"Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus.

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology ("SulfoSYS")-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the "-omics" approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics.

SulfoSYS (Sulfolobus Systems Biology): towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation.

SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. In Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bacteria or Eukarya, e.g. the unusual branched ED (Entner-Doudoroff) pathway, which is utilized for glucose degradation in S. solfataricus. This archaeal model organism of choice is a thermoacidophilic crenarchaeon that optimally grows at 80 degrees C (60-92 degrees C) and pH 2-4. In general, life at high temperature requires very efficient adaptation to temperature changes, which is most difficult to deal with for organisms, and it is unclear how biological networks can withstand and respond to such changes. This integrative project combines genomic, transcriptomic, proteomic and metabolomic, as well as kinetic and biochemical information. The final goal of SulfoSYS is the construction of a silicon cell model for this part of the living cell that will enable computation of the CCM network. In the present paper, we report on one of the first archaeal systems biology projects.

A novel trehalose synthesizing pathway in the hyperthermophilic Crenarchaeon Thermoproteus tenax: the unidirectional TreT pathway.

In the genome of the hyperthermophilic archaeon Thermoproteus tenax a gene (treS/P) encoding a protein with similarity to annotated trehalose phosphorylase (TreP), trehalose synthase (TreS) and more recently characterized trehalose glycosyltransferring synthase (TreT) was identified. The treS/P gene as well as an upstream located ORF of unknown function (orfY) were cloned, heterologously expressed in E. coli and purified. The enzymatic characterization of the putative TreS/P revealed TreT activity. However, contrary to the previously characterized reversible TreT from Thermococcus litoralis and Pyrococcus horikoshii, the T. tenax enzyme is unidirectional and catalyzes only the formation of trehalose from UDP (ADP)-glucose and glucose. The T. tenax enzyme differs from the reversible TreT of T. litoralis by its preference for UDP-glucose as co-substrate. Phylogenetic and comparative gene context analyses reveal a conserved organization of the unidirectional TreT and OrfY gene cluster that is present in many Archaea and a few Bacteria. In contrast, the reversible TreT pathway seems to be restricted to only a few archaeal (e.g. Thermococcales) and bacterial (Thermotogales) members. Here we present a new pathway exclusively involved in trehalose synthesis--the unidirectional TreT pathway--and discuss its physiological role as well as its phylogenetic distribution.

Volatilisation of metals and metalloids: an inherent feature of methanoarchaea?

As shown by recent studies, anaerobic members of Archaea and Bacteria are involved in processes that transform ionic species of metals and metalloids (arsenic, antimony, bismuth, selenium, tellurium and mercury) into volatile and mostly toxic derivatives (mainly methyl derivatives or hydrides). Since the fact that these transformations proceed in both environmental settings and in parts of the human body, we have to consider that these processes also interfere directly with human health. The diversity of the volatile derivatives produced and their emission rates were significantly higher in methanoarchaeal than in bacterial strains, which supports the pivotal role of methanoarchaea in transforming metals and metalloids (metal(loid)s) into their volatile derivatives. Compared with methanoarchaea, 14 anaerobic bacterial strains showed a significantly restricted spectrum of volatilised derivatives and mostly lower production rates of volatile bismuth and selenium derivatives. Since methanoarchaea isolated from the human gut (Methanosphaera stadtmanae, Methanobrevibacter smithii) showed a higher potential for metal(loid) derivatisation compared to bacterial gut isolates, we assume that methanoarchaea in the human gut are mainly responsible for the production of these volatile derivatives. The observation that trimethylbismuth ((CH(3))(3)Bi), the main volatile derivative of bismuth produced in human feces, inhibited growing cultures of Bacteroides thetaiotaomicron, a representative member of the human physiological gut flora, suggests that these volatiles exert their toxic effects on human health not only by direct interaction with host cells but also by disturbing the physiological gut microflora.