Johann M. Rohwer

Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data

Sucrose accumulation in developing sugar cane (Saccharum officinarum) is accompanied by a continuous synthesis and cleavage of sucrose in the storage tissues. Despite numerous studies, the factors affecting sucrose accumulation are still poorly understood, and no consistent pattern has emerged which pinpoints certain enzyme activities as important controlling steps. Here, we develop an approach based on pathway analysis and kinetic modelling to assess the biochemical control of sucrose accumulation and futile cycling in sugar cane. By using the concept of elementary flux modes, all possible routes of futile cycling of sucrose were enumerated in the metabolic system. The available kinetic data for the pathway enzymes were then collected and assembled in a kinetic model of sucrose accumulation in sugar cane culm tissue. Although no data were fitted, the model agreed well with independent experimental results: in no case was the difference between calculated and measured fluxes and concentrations greater than 2-fold. The model thus validated was then used to assess different enhancement strategies for increasing sucrose accumulation. First, the control coefficient of each enzyme in the system on futile cycling of sucrose was calculated. Secondly, the activities of those enzymes with the numerically largest control coefficients were varied over a 5-fold range to determine the effect on the degree of futile cycling, the conversion efficiency from hexoses into sucrose, and the net sucrose accumulation rate. In view of the modelling results, overexpression of the fructose or glucose transporter or the vacuolar sucrose import protein, as well as reduction of cytosolic neutral invertase levels, appear to be the most promising targets for genetic manipulation. This offers a more directed improvement strategy than cumbersome gene-by-gene manipulation. The kinetic model can be viewed and interrogated on the World Wide Web at http://jjj.biochem.sun.ac.za.

Modelling cellular systems with PySCeS

UNLABELLED The Python Simulator for Cellular Systems (PySCeS) is an extendable research tool for the numerical analysis and investigation of cellular systems. AVAILABILITY PySCeS is distributed as Open Source Software under the GNU General Public Licence and is available for download from http://pysces.sourceforge.net CONTACT bgoli@sun.ac.za.

Kinetic modelling of plant metabolic pathways

This paper provides a review of kinetic modelling of plant metabolic pathways as a tool for analysing their control and regulation. An overview of different modelling strategies is presented, starting with those approaches that only require a knowledge of the network stoichiometry; these are referred to as structural. Flux-balance analysis, metabolic flux analysis using isotope labelling, and elementary mode analysis are briefly mentioned as three representative examples. The main focus of this paper, however, is a discussion of kinetic modelling, which requires, in addition to the stoichiometry, a knowledge of the kinetic properties of the constituent pathway enzymes. The different types of kinetic modelling analysis, namely time-course simulation, steady-state analysis, and metabolic control analysis, are explained in some detail. An overview is presented of strategies for obtaining model parameters, as well as software tools available for simulation of such models. The kinetic modelling approach is exemplified with discussion of three models from the general plant physiology literature. With the aid of kinetic modelling it is possible to perform a control analysis of a plant metabolic system, to identify potential targets for biotechnological manipulation, as well as to ascertain the regulatory importance of different enzymes (including isoforms of the same enzyme) in a pathway. Finally, a framework is presented for extending metabolic models to the whole-plant scale by linking biochemical reactions with diffusion and advective flow through the phloem. Future challenges include explicit modelling of subcellular compartments, as well as the integration of kinetic models on the different levels of the cellular and organizational hierarchy.

Deoxyxylulose 5-phosphate synthase controls flux through the methylerythritol 4-phosphate pathway in Arabidopsis

1-Deoxyxylulose 5-phosphate synthase is the controlling enzyme of plastid isoprenoid precursor biosynthesis in Arabidopsis. The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.

Composite control of cell function: metabolic pathways behaving as single control units

This paper shows that under some conditions the control exerted by a part of a metabolic network (a pathway) on a flux or concentration in any other part can be described through a single (overall) control coefficient. This has the following implications: (i) the relative contributions of a pathway enzyme to the regulation of the pathway (output) flux and of any flux or concentration outside are identical; therefore, the control analysis of the pathway ‘in isolation’ allows one to determine the control exerted by any pathway enzyme on the rest of the cell by estimation of the control efficient of just one, arbitrarily chosen enzyme; (ii) the relative control of any two metabolic variables outside the pathway (measured as the ratio of the control coefficients over these two variables outside) is the same for all pathway enzymes. These properties allow one to substitute effectively a pathway by a single (super)reaction and make it possible to consider such a pathway as a metabolic unit within the cellular enzyme network.

Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model.

Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.

The logic of kinetic regulation in the thioredoxin system

BackgroundThe thioredoxin system consisting of NADP(H), thioredoxin reductase and thioredoxin provides reducing equivalents to a large and diverse array of cellular processes. Despite a great deal of information on the kinetics of individual thioredoxin-dependent reactions, the kinetic regulation of this system as an integrated whole is not known. We address this by using kinetic modeling to identify and describe kinetic behavioral motifs found within the system.ResultsAnalysis of a realistic computational model of the Escherichia coli thioredoxin system revealed several modes of kinetic regulation in the system. In keeping with published findings, the model showed that thioredoxin-dependent reactions were adaptable (i.e. changes to the thioredoxin system affected the kinetic profiles of these reactions). Further and in contrast to other systems-level descriptions, analysis of the model showed that apparently unrelated thioredoxin oxidation reactions can affect each other via their combined effects on the thioredoxin redox cycle. However, the scale of these effects depended on the kinetics of the individual thioredoxin oxidation reactions with some reactions more sensitive to changes in the thioredoxin cycle and others, such as the Tpx-dependent reduction of hydrogen peroxide, less sensitive to these changes. The coupling of the thioredoxin and Tpx redox cycles also allowed for ultrasensitive changes in the thioredoxin concentration in response to changes in the thioredoxin reductase concentration. We were able to describe the kinetic mechanisms underlying these behaviors precisely with analytical solutions and core models.ConclusionsUsing kinetic modeling we have revealed the logic that underlies the functional organization and kinetic behavior of the thioredoxin system. The thioredoxin redox cycle and associated reactions allows for a system that is adaptable, interconnected and able to display differential sensitivities to changes in this redox cycle. This work provides a theoretical, systems-biological basis for an experimental analysis of the thioredoxin system and its associated reactions.

Enzymes or redox couples? The kinetics of thioredoxin and glutaredoxin reactions in a systems biology context.

Systems biology approaches, such as kinetic modelling, could provide valuable insights into how thioredoxins, glutaredoxins and peroxiredoxins (here collectively called redoxins), and the systems that reduce these molecules are regulated. However, it is not clear whether redoxins should be described as redox couples (with redox potentials) or as enzymes (with Michaelis-Menten parameters) in such approaches. We show that in complete redoxin systems, redoxin substrate saturation and other purported enzymatic behaviours result from limitations in the redoxin redox cycles in these systems. Michaelis-Menten parameters are therefore inappropriate descriptors of redoxin activity; data from redoxin kinetic experiments should rather be interpreted in terms of the complete system of reactions under study. These findings were confirmed by fitting kinetic models of the thioredoxin and glutaredoxin systems to in vitro datasets. This systems approach clarifies the inconsistencies with the descriptions of redoxins and emphasizes the roles of redoxin systems in redox regulation.

From steady-state to synchronized yeast glycolytic oscillations I: Model construction

An existing detailed kinetic model for the steady‐state behavior of yeast glycolysis was tested for its ability to simulate dynamic behavior. Using a small subset of experimental data, the original model was adapted by adjusting its parameter values in three optimization steps. Only small adaptations to the original model were required for realistic simulation of experimental data for limit‐cycle oscillations. The greatest changes were required for parameter values for the phosphofructokinase reaction. The importance of ATP for the oscillatory mechanism and NAD(H) for inter‐and intra‐cellular communications and synchronization was evident in the optimization steps and simulation experiments. In an accompanying paper [du Preez F et al. (2012) FEBS J279, 2823–2836], we validate the model for a wide variety of experiments on oscillatory yeast cells. The results are important for re‐use of detailed kinetic models in modular modeling approaches and for approaches such as that used in the Silicon Cell initiative.

Supply–Demand Analysis: A Framework for Exploring the Regulatory Design of Metabolism

The living cell can be thought of as a collection of linked chemical factories, a molecular economy in which the principles of supply and demand obtain. Supply-demand analysis is a framework for exploring and gaining an understanding of metabolic regulation, both theoretically and experimentally, where regulatory performance is measured in terms of flux control and homeostatic maintenance of metabolite concentrations. It is based on a metabolic control analysis of a supply-demand system in steady state in which the degree of flux and concentration control by the supply and demand blocks is related to their local properties, which are quantified as the elasticities of supply and demand. These elasticities can be visualized as the slopes of the log-log rate characteristics of supply and demand. Rate characteristics not only provide insight about system behavior around the steady state but can also be expanded to provide a view of the behavior of the system over a wide range of concentrations of the metabolic intermediate that links the supply and the demand. The theoretical and experimental results of supply-demand analysis paint a picture of the regulatory design of metabolic systems that differs radically from what can be called the classical view of metabolic regulation, which generally explains the role of regulatory mechanisms only in terms of the supply, completely ignoring the demand. Supply-demand analysis has recently been generalized into a computational tool that can be used to study the regulatory behavior of kinetic models of metabolic systems up to genome-scale.