Transient physical association between enzymes appears to be a cardinal feature of metabolic systems, yet the purpose of this metabolic organisation remains enigmatic. It is generally assumed that ...substrate channelling occurs in these complexes. However, there is a lack of information concerning the mechanisms and extent of substrate channelling and confusion regarding the consequences of substrate channelling. In this review, we outline recent advances in the structural characterisation of enzyme assemblies and integrate this with new insights from reaction-diffusion modelling and synthetic biology to clarify the mechanistic and functional significance of the phenomenon.
Identifying the correct subcellular locations for all enzymes and metabolites in plant metabolic networks is a major challenge, but is critically important for the success of the new generation of ...large-scale metabolic models that are driving a network-level appreciation of metabolic behavior. Even though the subcellular compartmentation of many central metabolic processes is thought to be well understood, recent gene-by-gene studies have revealed several unexpected enzyme localizations. Metabolite transport between subcellular compartments is crucial because it fundamentally affects the metabolic network structure. Although new metabolite transporters are being steadily identified, modeling work suggests that we have barely scratched the surface of the catalog of intracellular metabolite transporter proteins. In addition to compartmentation among organelles, it is increasingly apparent that microcompartment formation via the interactions of enzyme groups with intracellular membranes, the cytoskeleton, or other proteins is an important regulatory mechanism. In particular, this mechanism can promote metabolite channeling within the metabolic microcompartment, which can help control reaction specificity as well as dictate flux routes through the network. This has clear relevance for both synthetic biology in general and the engineering of plant metabolic networks in particular.
Key aspects of leaf mitochondrial metabolism in the light remain unresolved. For example, there is debate about the relative importance of exporting reducing equivalents from mitochondria for the ...peroxisomal steps of photorespiration versus oxidation of NADH to generate ATP by oxidative phosphorylation. Here, we address this and explore energetic coupling between organelles in the light using a diel flux balance analysis model. The model included more than 600 reactions of central metabolism with full stoichiometric accounting of energy production and consumption. Different scenarios of energy availability (light intensity) and demand (source leaf versus a growing leaf) were considered, and the model was constrained by the nonlinear relationship between light and CO
assimilation rate. The analysis demonstrated that the chloroplast can theoretically generate sufficient ATP to satisfy the energy requirements of the rest of the cell in addition to its own. However, this requires unrealistic high light use efficiency and, in practice, the availability of chloroplast-derived ATP is limited by chloroplast energy dissipation systems, such as nonphotochemical quenching, and the capacity of the chloroplast ATP export shuttles. Given these limitations, substantial mitochondrial ATP synthesis is required to fulfill cytosolic ATP requirements, with only minimal, or zero, export of mitochondrial reducing equivalents. The analysis also revealed the importance of exporting reducing equivalents from chloroplasts to sustain photorespiration. Hence, the chloroplast malate valve and triose phosphate-3-phosphoglycerate shuttle are predicted to have important metabolic roles, in addition to their more commonly discussed contribution to the avoidance of photooxidative stress.
Reactive oxygen species (ROS) production increases in plants under stress. ROS can damage cellular components, but they can also act in signal transduction to help the cell counteract the oxidative ...damage in the stressed compartment. H
2O
2 might induce a general stress response, but it does not have the required specificity to selectively regulate nuclear genes required for dealing with localized stress, e.g. in chloroplasts or mitochondria. Here we argue that peptides deriving from proteolytic breakdown of oxidatively damaged proteins have the requisite specificity to act as secondary ROS messengers and regulate source-specific genes and in this way contribute to retrograde ROS signalling during oxidative stress. Likewise, unmodified peptides deriving from the breakdown of redundant proteins could help coordinate organellar and nuclear gene expression.
Acetylation of the ε-amino group of lysine (Lys) is a reversible posttranslational modification recently discovered to be widespread, occurring on proteins outside the nucleus, in most subcellular ...locations in mammalian cells. Almost nothing is known about this modification in plants beyond the well-studied acetylation of historie proteins in the nucleus. Here, we report that Lys acetylation in plants also occurs on organellar and cytosolic proteins. We identified 91 Lys-acetylated sites on 74 proteins of diverse functional classes. Furthermore, our study suggests that Lys acetylation may be an important posttranslational modification in the chloroplast, since four Calvin cycle enzymes were acetylated. The plastid-encoded large subunit of Rubisco stands out because of the large number of acetylated sites occurring at important Lys residues that are involved in Rubisco tertiary structure formation and catalytic function. Using the human recombinant deacetylase sirtuin 3, it was demonstrated that Lys deacetylation significantly affects Rubisco activity as well as the activities of other central metabolic enzymes, such as the Calvin cycle enzyme phosphoglycerate kinase, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, and the tricarboxylic acid cycle enzyme malate dehydrogenase. Our results demonstrate that Lys acetylation also occurs on proteins outside the nucleus in Arabidopsis (Arabidopsis thaliana) and that Lys acetylation could be important in the regulation of key metabolic enzymes.
Crassulacean acid metabolism (CAM) evolved in arid environments as a water-saving alternative to C
photosynthesis. There is great interest in engineering more drought-resistant crops by introducing ...CAM into C
plants. However, it is unknown whether full CAM or alternative water-saving modes would be more productive in the environments typically experienced by C
crops. To study the effect of temperature and relative humidity on plant metabolism in the context of water saving, we coupled a time-resolved diel (based on a 24-h day-night cycle) model of leaf metabolism to an environment-dependent gas-exchange model. This combined model allowed us to study the emergence of CAM as a trade-off between leaf productivity and water saving. We show that vacuolar storage capacity in the leaf is a major determinant of the extent of CAM. Moreover, our model identified an alternative CAM cycle involving mitochondrial isocitrate dehydrogenase as a potential contributor to initial carbon fixation at night. Simulations across a range of environmental conditions show that the water-saving potential of CAM strongly depends on the daytime weather conditions and that the additional water-saving effect of carbon fixation by isocitrate dehydrogenase can reach 11% total water saving for the conditions tested.
The properties of a cpYFP circularly permuted YFP (yellow fluorescent protein) reported to act as a superoxide sensor have been re-examined in Arabidopsis mitochondria. We have found that the probe ...has high pH sensitivity and that dynamics in the cpYFP signal disappeared when the matrix pH was clamped by nigericin. In contrast, genetic and pharmacological manipulation of matrix superoxide had no detectable effect on the cpYFP signal. These findings question the existence of superoxide flashes in mitochondria.
Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway ...fluxes. Experimental evidence supporting their existence in vivo remains fragmentary. In the present study, we test binary interactions of the proteins constituting the plant tricarboxylic acid (TCA) cycle. We integrate (semi-)quantitative results from affinity purification-mass spectrometry, split-luciferase and yeast-two-hybrid assays to generate a single reliability score for assessing protein-protein interactions. By this approach, we identify 158 interactions including those between catalytic subunits of sequential enzymes and between subunits of enzymes mediating non-adjacent reactions. We reveal channelling of citrate and fumarate in isolated potato mitochondria by isotope dilution experiments. These results provide evidence for a functional TCA cycle metabolon in plants, which we discuss in the context of contemporary understanding of this pathway in other kingdoms.
The tricarboxylic acid (TCA) cycle is a crucial component of respiratory metabolism in both photosynthetic and heterotrophic plant organs. All of the major genes of the tomato TCA cycle have been ...cloned recently, allowing the generation of a suite of transgenic plants in which the majority of the enzymes in the pathway are progressively decreased. Investigations of these plants have provided an almost complete view of the distribution of control in this important pathway. Our studies suggest that citrate synthase, aconitase, isocitrate dehydrogenase, succinyl CoA ligase, succinate dehydrogenase, fumarase and malate dehydrogenase have control coefficients flux for respiration of −0.4, 0.964, −0.123, 0.0008, 0.289, 0.601 and 1.76, respectively; while 2‐oxoglutarate dehydrogenase is estimated to have a control coefficient of 0.786 in potato tubers. These results thus indicate that the control of this pathway is distributed among malate dehydrogenase, aconitase, fumarase, succinate dehydrogenase and 2‐oxoglutarate dehydrogenase. The unusual distribution of control estimated here is consistent with specific non‐cyclic flux mode and cytosolic bypasses that operate in illuminated leaves. These observations are discussed in the context of known regulatory properties of the enzymes and some illustrative examples of how the pathway responds to environmental change are given.
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