Bioinformatics, cellular flows, and calculation.
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Algorithms↗
Biomedical subjects
J L Snoep
Publications and source records attributed to J L Snoep.
Time dependent responses of glycolytic intermediates in a detailed glycolytic model of Lactococcus lactis during glucose run-out experiments.
Glucose addition and subsequent run-out experiments were compared to simulations with a detailed glycolytic model of Lactococcus lactis. The model was constructed largely on bases of enzyme kinetic data taken from literature and not adjusted for the specific simulations shown here. Upon glucose depletion a rapid increase in PEP, inorganic phosphate and a gradual decrease in fructose 1,6-bisphosphate (FBP) were measured and predicted by simulation. The dynamic changes in these and other intermediate concentrations as measured in the experiments were well predicted by the kinetic model.
Adenosine Triphosphate↗
How to distinguish between the vacuum cleaner and flippase mechanisms of the lmrA multi-drug transporter in Lactococcus lactis.
A numerical model of the LmrA multi-drug transport system of Lactococcus lactis is used to explore the possibility of distinguishing experimentally between two putative transport mechanisms, i.e., the vacuum-cleaner and the flippase mechanisms. This comparative model also serves as an example of numerical simulation with the scripting language Python and its scientific add-on Scipy.
Bacterial Proteins↗
Java Web Simulation (JWS); a web based database of kinetic models.
Software to make a database of kinetic models accessible via the internet has been developed and a core database has been set up at http://jjj.biochem.sun.ac.za/. This repository of models, available to everyone with internet access, opens a whole new way in which we can make our models public. Via the database, a user can change enzyme parameters and run time simulations or steady state analyses. The interface is user friendly and no additional software is necessary. The database currently contains 10 models, but since the generation of the program code to include new models has largely been automated the addition of new models is straightforward and people are invited to submit their models to be included in the database.
Computer Simulation↗
Experimental supply-demand analysis of anaerobic yeast energy metabolism.
Experimental supply-demand analysis of yeast fermentative energy metabolism shows that control of the glycolytic flux is shared between supply and demand. In glucose limited chemostat cultures the supply block was modulated in a dilution rate change and demand block via a benzoic acid titration. Under these conditions the supply block had a flux control of 0.90 and the demand block a flux control of 0.10.
Adenosine Diphosphate↗
Control of glycolytic dynamics by hexose transport in Saccharomyces cerevisiae.
It is becoming accepted that steady-state fluxes are not necessarily controlled by single rate-limiting steps. This leaves open the issue whether cellular dynamics are controlled by single pacemaker enzymes, as has often been proposed. This paper shows that yeast sugar transport has substantial but not complete control of the frequency of glycolytic oscillations. Addition of maltose, a competitive inhibitor of glucose transport, reduced both average glucose consumption flux and frequency of glycolytic oscillations. Assuming a single kinetic component and a symmetrical carrier, a frequency control coefficient of between 0.4 and 0.6 and an average-flux control coefficient of between 0.6 and 0.9 were calculated for hexose transport activity. In a second approach, mannose was used as the carbon and free-energy source, and the dependencies on the extracellular mannose concentration of the transport activity, of the frequency of oscillations, and of the average flux were compared. In this case the frequency control coefficient and the average-flux control coefficient of hexose transport activity amounted to 0.7 and 0.9, respectively. From these results, we conclude that 1) transport is highly important for the dynamics of glycolysis, 2) most but not all control resides in glucose transport, and 3) there should at least be one step other than transport with substantial control.
Biological Transport, Active↗
Glucose and the ATP paradox in yeast.
A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis. We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis. With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route.
Adenosine Triphosphate↗
Transduction of intracellular and intercellular dynamics in yeast glycolytic oscillations.
Under certain well-defined conditions, a population of yeast cells exhibits glycolytic oscillations that synchronize through intercellular acetaldehyde. This implies that the dynamic phenomenon of the oscillation propagates within and between cells. We here develop a method to establish by which route dynamics propagate through a biological reaction network. Application of the method to yeast demonstrates how the oscillations and the synchronization signal can be transduced. That transduction is not so much through the backbone of glycolysis, as via the Gibbs energy and redox coenzyme couples (ATP/ADP, and NADH/NAD), and via both intra- and intercellular acetaldehyde.
Acetaldehyde↗
Gearing up for a quantitative understanding of cell (dys)function.
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Animals↗
Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry.
This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured. In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.
Enzymes↗
Construction and characterization of an effector strain of Streptococcus mutans for replacement therapy of dental caries.
An effector strain has been constructed for use in the replacement therapy of dental caries. Recombinant DNA methods were used to make the Streptococcus mutans supercolonizing strain, JH1140, lactate dehydrogenase deficient by deleting virtually all of the ldh open reading frame (ORF). To compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the adhB ORF from Zymomonas mobilis in place of the deleted ldh ORF. The resulting clone, BCS3-L1, was found to produce no detectable lactic acid during growth on a variety of carbon sources, and it produced significantly less total acid due to its increased production of ethanol and acetoin. BCS3-L1 was significantly less cariogenic than JH1140 in both gnotobiotic- and conventional-rodent models. It colonized the teeth of conventional rats as well as JH1140 in both aggressive-displacement and preemptive-colonization models. No gross or microscopic abnormalities of major organs were associated with oral colonization of rats with BCS3-L1 for 6 months. Acid-producing revertants of BCS3-L1 were not observed in samples taken from infected animals (reversion frequency, <10(-3)) or by screening cultures grown in vitro, where no revertants were observed among 10(5) colonies examined on pH indicator medium. The reduced pathogenic potential of BCS3-L1, its strong colonization potential, and its genetic stability suggest that this strain is well suited to serve as an effector strain in the replacement therapy of dental caries in humans.
Animals↗
Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a new, secreted metabolite serving as a temporary redox sink.
Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain alpha-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain alpha-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the alpha-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of alpha-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD(+) from the NADH produced by the branched-chain alpha-keto acid dehydrogenase complex was required for complete conversion of alpha-ketoisocaproate. Interestingly, during the conversion of the branched-chain alpha-keto acids an intermediate was always detected extracellularly. With alpha-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1, 1-dihydroxy-4-methyl-2-pentanone. This reduced form of alpha-ketoisocaproic acid was found to serve as a temporary redox sink.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Live control of the living cell.
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Animals↗
Extensive regulation compromises the extent to which DNA gyrase controls DNA supercoiling and growth rate of Escherichia coli.
As DNA gyrase is the only enzyme to supercoil DNA actively, we address here the question of whether it does play the expected dominant role in controlling the level of DNA supercoiling and growth rate in Escherichia coli. We modulated the expression of DNA gyrase around its wild-type level, and measured the effect on plasmid supercoiling and growth rate. As both the activity and the transcription rate of DNA gyrase are sensitive to DNA supercoiling we distinguish two types of control (with control defined as the percentage change observed on a 1% modulation of a parameter). The first type of control, here named inherent control, quantifies the effect of a sustained modulation of the transcription rate of gyrase. At its wild-type expression level this inherent control exerted by DNA gyrase on growth rate was very low, i.e. c mu/gyrase = 0.05 - 0.00, as was the inherent control on DNA supercoiling, c aLK/gyrase = 0.2. The second type of control, here named global control, quantifies the effect of a change in gyrase activity whilst allowing the cell to respond by readjusting gyrase transcription. Both types of control are linked via the sensitivity of gyrase transcription to DNA supercoiling, as determined from the inherent control by gyrase of the gyrase promoter activity using a chromosomal gyrB:lacZ fusion. As expected, the latter control was negative, but small, i.e. c gyr promoter/gyrase=-0.3. The global control by gyrase of active linking number was 0.1. These results show that although gyrase is an essential enzyme it does not have a high control, on either growth rate or DNA supercoiling. Homeostatic regulation of physiological DNA structure appears to dominate. At low degrees of DNA supercoiling, the control by DNA gyrase and by the other topoisomerases is much stronger.
Adenosine Diphosphate↗
Catabolism of branched-chain alpha-keto acids in Enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route.
Genes encoding a branched-chain alpha-keto acid dehydrogenase from Enterococcus faecalis 10C1, E1alpha (bkdA), E1beta (bkdB), E2 (bkdC), and E3 (bkdD), were found to reside in the gene cluster ptb-buk-bkdDABC. The predicted products of ptb and buk exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from Clostridium acetobutylicum. Activity and redox properties of the purified recombinant enzyme encoded by bkdD indicate that E. faecalis has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the ptb gene product expressed in Escherichia coli was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of alpha-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that alpha-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the ptb and buk gene products with the concomitant phosphorylation of ADP. In contrast, an E. faecalis bkd mutant constructed by disruption of the bkdA gene did not benefit from having alpha-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the bkd gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain alpha-keto acids that was previously unidentified in E. faecalis.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli.
Escherichia coli MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT </= 1%). A simultaneous flux from pyruvate through both PFL and the pyruvate dehydrogenase complex (PDHc) was observed. In anaerobic cultures with fumarate or nitrate as an electron acceptor, a significant flux through the PDHc was calculated on the basis of the redox balance, the measured products, and the known biochemistry. This result calls into question the common assumption that the complex cannot be active under these conditions. In vitro activity measurements of PDHc showed that the cellular content of the enzyme varied with the internal redox state and revealed an activity for dissolved oxygen tension of below 1%. Whereas Western blots showed that the E3 subunit of PDHc (dihydrolipoamide dehydrogenase) did not vary to a large extent under the conditions tested, the E2 subunit (dihydrolipoamide acetyltransferase) amount followed the trend that was found for the in vitro PDHc activity. From this it is concluded that regulation of the PDHc is exerted at the E1/E2 operon (aceEF). We propose that the external redox state (measured as the midpoint potentials of those terminal acceptors with which the cell has sufficient capacity to react) is reflected by the internal redox state. The latter may subsequently govern both the expression and the activity of the two pyruvate-catabolizing enzymes.
Acetyltransferases↗
Regulation of energy source metabolism in streptococci.
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Adaptation, Biological↗
DNA supercoiling depends on the phosphorylation potential in Escherichia coli.
ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ ADP ratio was reduced by a shift to anaerobic conditions, by addition of a protonophore (dinitrophenol) or by potassium cyanide addition, DNA supercoiling decreased similarly with the ATP/ADP ratio. The experiments were performed under well-defined conditions, where oxidative phosphorylation was the dominant route for ATP synthesis, i.e. using a minimal salts medium with succinate as the sole free-energy and carbon source, and in the presence or absence of ammonia as the nitrogen source. The results of the different experiments were consistent with a single linear relationship between the log(ATP/ADP) and the change in linking number. The dependence of DNA supercoiling on the ATP/ADP ratio was not influenced by inhibitors of transcription or translation. Because the ATP/ADP ratio was modulated in different ways, the unique relationship suggests coupling between the phosphorylation potential and DNA supercoiling. This was most probably mediated by the DNA gyrase, independent of topoisomerase I or transcription.
Adenosine Diphosphate↗