Umeå University's logo

Nucleoside mono-, di- and triphosphates (NMP, NDP, and NTP) and their deoxy-counterparts (dNMP, dNDP, dNTP) are involved in energy metabolism and are the building blocks of RNA and DNA, respectively. The production of NTP and dNTP is carried out by several NMP kinases (NMPK) and NDP kinases (NDPK). All NMPKs are fully reversible and use defined Mg-free and Mg-complexed nucleotides in both directions of their reactions, with Mg2+ controlling the ratios of Mg-free and Mg-complexed reactants. Their activities are driven by adenylates produced by adenylate kinase which controls the direction of NMPK and NDPK reactions, depending on the energy status of a cell. This enzymatic machinery is localized in the cytosol, mitochondria, and plastids, i.e. compartments with high energy budgets and where (except for cytosol) RNA and DNA synthesis occur. Apparent equilibrium constants of NMPKs, based on total nucleotide contents, are [Mg2+]-dependent. This allows for an indirect estimation of internal [Mg2+], which constitutes a signal of the energetic status of a given tissue/cell/compartment. Adenylates contribute the most to this Mg2+-signal, followed by uridylates, guanylates, and cytidylates, with deoxynucleotides’ contribution deemed negligible. A method to quantify the Mg2+-signal, using nucleotide datasets, is discussed.

Plant glycerate kinase (GK) was previously considered an exclusively chloroplastic enzyme of the glycolate pathway (photorespiration), and its sole predicted role was to return most of the glycolate-derived carbon (as glycerate) to the Calvin cycle. However, recent discovery of cytosolic GK revealed metabolic links for glycerate to other processes. Although GK was initially proposed as being solely regulated by substrate availability, subsequent discoveries of its redox regulation and the light involvement in the production of chloroplastic and cytosolic GK isoforms have indicated a more refined regulation of the pathways of glycerate conversion. Here, we re-evaluate the importance of GK and emphasize its multifaceted role in plants. Thus, GK can be a major player in several branches of primary metabolism, including the glycolate pathway, gluconeogenesis, glycolysis, and C4 metabolism. In addition, recently, the chloroplastic (but not cytosolic) GK isoform was implicated as part of a light-dependent plant immune response to pathogen attack. The origins of glycerate are also discussed here; it is produced in several cell compartments and undergoes huge fluctuations depending on light/dark conditions. The recent discovery of the vacuolar glycerate transporter adds yet another layer to our understanding of glycerate transport/metabolism and that of other two- and three-carbon metabolites.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) catalyzes a reversible reaction, producing UDPG, which serves as an essential precursor for hundreds of glycosyltransferases in all organisms. In this study, activities of purified UGPases from sugarcane and barley were found to be reversibly redox modulated in vitro through oxidation by hydrogen peroxide or oxidized glutathione (GSSG) and through reduction by dithiothreitol or glutathione. Generally, while oxidative treatment decreased UGPase activity, a subsequent reduction restored the activity. The oxidized enzyme had increased K_m values with substrates, especially pyrophosphate. The increased K_m values were also observed, regardless of redox status, for UGPase cysteine mutants (Cys102Ser and Cys99Ser for sugarcane and barley UGPases, respectively). However, activities and substrate affinities (K_ms) of sugarcane Cys102Ser mutant, but not barley Cys99Ser, were still prone to redox modulation. The data suggest that plant UGPase is subject to redox control primarily via changes in the redox status of a single cysteine. Other cysteines may also, to some extent, contribute to UGPase redox status, as seen for sugarcane enzymes. The results are discussed with respect to earlier reported details of redox modulation of eukaryotic UGPases and regarding the structure/function properties of these proteins.

Free magnesium (Mg²⁺) represents a powerful signal arising from interconversions of adenylates (ATP, ADP and AMP). This is a consequence of the involvement of adenylate kinase (AK) which equilibrates adenylates and uses defined species of Mg-complexed and Mg-free adenylates in both directions of its reaction. However, cells contain also other reversible Mg²⁺-dependent enzymes that equilibrate non-adenylate nucleotides (uridylates, cytidylates and guanylates), i.e. nucleoside monophosphate kinases (NMPKs) and nucleoside diphosphate kinase (NDPK). Here, we propose that AK activity is tightly coupled to activities of NMPK and NDPK, linking adenylate equilibrium to equilibria of other nucleotides, and with [Mg²⁺] controlling the ratios of Mg-chelated and Mg-free nucleotides. This coupling establishes main hubs for adenylate-driven equilibration of non-adenylate nucleotides, with [Mg²⁺] acting as signal arising from all nucleotides rather than adenylates only. Further consequences involve an overall adenylate control of UTP-, GTP- and CTP-dependent pathways and the availability of substrates for RNA and DNA synthesis.

General structure of metabolism includes the reproduction of catalysts that govern metabolism. In this structure, the system becomes autopoietic in the sense of Maturana and Varela, and it is closed to efficient causation as defined by Robert Rosen. The autopoietic maintenance and operation of the catalysts takes place via the set of free nucleotides while the synthesis of catalysts occurs via the information encoded by the set of nucleotides arranged in polymers of RNA and DNA. Both energy charge and genetic information use the components of the same pool of nucleoside triphosphates, which is equilibrated by thermodynamic buffering enzymes such as nucleoside diphosphate kinase and adenylate kinase. This occurs in a way that the system becomes internally stable and metabolically closed, which initially could be realized at the level of ribozymes catalyzing basic metabolic reactions as well as own reproduction. The function of ATP, GTP, UTP, and CTP is dual, as these species participate both in the general metabolism as free nucleotides and in the transfer of genetic information via covalent polymerization to nucleic acids. The changes in their pools directly impact both bioenergetic pathways and nucleic acid turnover. Here we outline the concept of metabolic closure of biosystems grounded in the dual function of nucleotide coenzymes that serve both as energetic and informational molecules and through this duality generate the autopoietic performance and the ability for codepoietic evolutionary transformations of living systems starting from the emergence of prebiotic systems.

UDP-glucose pyrophosphorylase (UGPase) carries a freely reversible reaction, using glucose-1-P and UTP to produce UDP-glucose (UDPG) and pyrophosphate (PP_i ), with UDPG being essential for glycosylation reactions in all organisms including, e.g., synthesis of sucrose, cellulose and glycoproteins. In the present study, we found that free magnesium (Mg²⁺) had profound effects on the reverse reaction of purified barley UGPase, and was absolutely required for its activity, with an apparent K_m of 0.13 mM. More detailed analyses with varied concentrations of MgPP_i allowed us to conclude that it is the MgPP_i complex which serves as true substrate for UGPase in its reverse reaction, with an apparent K_m of 0.06 mM. Free PPi was an inhibitor in this reaction. Given the key role of PP_i in the UGPase reaction, we have also tested possible effects of phosphonates, which are analogs of PP_i and phosphate (P_i ). Clodronate and etidronate (PP_i analogs) had little or no effect on UGPase activity, whereas fosetyl-Al (P_i analog), a known fungicide, acted as effective near-competitive inhibitor versus PP_i, with K_i of 0.15 mM. The data are discussed with respect to the role of magnesium in the UGPase reaction and elucidating the use of inhibitors in studies on cellular function of UGPase and related enzymes.

Free magnesium (Mg²⁺) is a signal of the adenylate (ATP+ADP+AMP) status in the cells. It results from the equilibrium of adenylate kinase (AK), which uses Mg-chelated and Mgfree adenylates as substrates in both directions of its reaction. The AK-mediated primary control of intracellular [Mg²⁺] is finely interwoven with the operation of membrane-bound adenylateand Mg²⁺-translocators, which in a given compartment control the supply of free adenylates and Mg²⁺ for the AK-mediated equilibration. As a result, [Mg²⁺] itself varies both between and within the compartments, depending on their energetic status and environmental clues. Other key nucleotide-utilizing/producing enzymes (e.g., nucleoside diphosphate kinase) may also be involved in fine-tuning of the intracellular [Mg²⁺]. Changes in [Mg²⁺] regulate activities of myriads of Mgutilizing/ requiring enzymes, affecting metabolism under both normal and stress conditions, and impacting photosynthetic performance, respiration, phloem loading and other processes. In compartments controlled by AK equilibrium (cytosol, chloroplasts, mitochondria, nucleus), the intracellular [Mg²⁺] can be calculated from total adenylate contents, based on the dependence of the apparent equilibrium constant of AK on [Mg²⁺]. Magnesium signaling, reflecting cellular adenylate status, is likely widespread in all eukaryotic and prokaryotic organisms, due simply to the omnipresent nature of AK and to its involvement in adenylate equilibration.

In the conditions of [Mg²⁺] elevation that occur, in particular, under low oxygen stress and are the consequence of the decrease in [ATP] and increase in [ADP] and [AMP], pyrophosphate (PPi) can function as an alternative energy currency in plant cells. In addition to its production by various metabolic pathways, PPi can be synthesized in the combined reactions of pyruvate, phosphate dikinase (PPDK) and pyruvate kinase (PK) by so-called PK/PPDK substrate cycle, and in the reverse reaction of membrane-bound H⁺-pyrophosphatase, which uses the energy of electrochemical gradients generated on tonoplast and plasma membrane. The PPi can then be consumed in its active forms of MgPPi and Mg₂PPi by PPi-utilizing enzymes, which require an elevated [Mg²⁺]. This ensures a continuous operation of glycolysis in the conditions of suppressed ATP synthesis, keeping metabolism energy efficient and less dependent on ATP.

Plant polysaccharides (cellulose, hemicellulose, pectin, starch) are either direct (i.e. leaf starch) or indirect products of photosynthesis, and they belong to the most abundant organic compounds in nature. Although each of these polymers is made by a specific enzymatic machinery, frequently in different cell locations, details of their synthesis share certain common features. Thus, the production of these polysaccharides is preceded by the formation of nucleotide sugars catalyzed by fully reversible reactions of various enzymes, mostly pyrophosphorylases. These 'buffering' enzymes are, generally, quite active and operate close to equilibrium. The nucleotide sugars are then used as substrates for irreversible reactions of various polysaccharide-synthesizing glycosyltransferases ('engine' enzymes), e.g. plastidial starch synthases, or plasma membrane-bound cellulose synthase and callose synthase, or ER/Golgi-located variety of glycosyltransferases forming hemicellulose and pectin backbones. Alternatively, the irreversible step might also be provided by a carrier transporting a given immediate precursor across a membrane. Here, we argue that local equilibria, established within metabolic pathways and cycles resulting in polysaccharide production, bring stability to the system via the arrangement of a flexible supply of nucleotide sugars. This metabolic system is itself under control of adenylate kinase and nucleoside-diphosphate kinase, which determine the availability of nucleotides (adenylates, uridylates, guanylates and cytidylates) and Mg2+, the latter serving as a feedback signal from the nucleotide metabolome. Under these conditions, the supply of nucleotide sugars to engine enzymes is stable and constant, and the metabolic process becomes optimized in its load and consumption, making the system steady and self-regulated.

The equilibria of coenzyme nucleotides and substrates established in plant cells generate simple rules that govern the plant metabolome and provide optimal conditions for the non-equilibrium fluxes of major metabolic processes such as ATP synthesis, CO2 fixation, and mitochondrial respiration. Fast and abundant enzymes, such as adenylate kinase, carbonic anhydrase or malate dehydrogenase, provide constant substrate flux for these processes. These "buffering" enzymes follow the Michaelis-Menten (MM) kinetics and operate near equilibrium. The non-equilibrium "engine" enzymes, such as ATP synthase, Rubisco or the respiratory complexes, follow the modified version of MM kinetics due to their high concentration and low concentration of their substrates. The equilibrium reactions serve as control gates for the non-equilibrium flux through the engine enzymes establishing the balance of the fluxes of load and consumption of metabolic components. Under the coordinated operation of buffering and engine enzymes, the concentrations of free and Mg-bound adenylates and of free Mg2+ are set, serving as feedback signals from the adenylate metabolome. Those are linked to various cell energetics parameters, including membrane potentials. Also, internal levels of reduced and oxidized pyridine nucleotides are established in the coordinated operation of malate dehydrogenase and respiratory components, with proton concentration as a feedback from pyridine nucleotide pools. Non-coupled pathways of respiration serve to equilibrate the levels of pyridine nucleotides, adenylates, and as a pH stat. This stable non-equilibrium organizes the fluxes of energy spatially and temporally, controlling the rates of major metabolic fluxes that follow thermodynamically and kinetically defined computational principles.