In Vitro Skeletal Muscle Model of PGM1 Deficiency Reveals Altered Energy Homeostasis.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

Plastidic phosphoglucomutase and ADP-glucose pyrophosphorylase mutants impair starch synthesis in rice pollen grains and cause male sterility.

To elucidate the starch synthesis pathway and the role of this reserve in rice pollen, we characterized mutations in the plastidic phosphoglucomutase, OspPGM, and the plastidic large subunit of ADP-glucose (ADP-Glc) pyrophosphorylase, OsAGPL4 Both genes were up-regulated in maturing pollen, a stage when starch begins to accumulate. Progeny analysis of self-pollinated heterozygous lines carrying the OspPGM mutant alleles, osppgm-1 and osppgm-2, or the OsAGPL4 mutant allele, osagpl4-1, as well as reciprocal crosses between the wild type (WT) and heterozygotes revealed that loss of OspPGM or OsAGPL4 caused male sterility, with the former condition rescued by the introduction of the WT OspPGM gene. While iodine staining and transmission electron microscopy analyses of pollen grains from homozygous osppgm-1 lines produced by anther culture confirmed the starch null phenotype, pollen from homozygous osagpl4 mutant lines, osagpl4-2 and osagpl4-3, generated by the CRISPR/Cas system, accumulated small amounts of starch which were sufficient to produce viable seed. Such osagpl4 mutant pollen, however, was unable to compete against WT pollen successfully, validating the important role of this reserve in fertilization. Our results demonstrate that starch is mainly polymerized from ADP-Glc synthesized from plastidic hexose phosphates in rice pollen and that starch is an essential requirement for successful fertilization in rice.

Loss of cytosolic phosphoglucomutase compromises gametophyte development in Arabidopsis.

Cytosolic phosphoglucomutase (cPGM) interconverts glucose-6-phosphate and glucose-1-phosphate and is a key enzyme of central metabolism. In this study, we show that Arabidopsis (Arabidopsis thaliana) has two cPGM genes (PGM2 and PGM3) encoding proteins with high sequence similarity and redundant functions. Whereas pgm2 and pgm3 single mutants were undistinguishable from the wild type, loss of both PGM2 and PGM3 severely impaired male and female gametophyte function. Double mutant pollen completed development but failed to germinate. Double mutant ovules also developed normally, but approximately half remained unfertilized 2 d after pollination. We attribute these phenotypes to an inability to effectively distribute carbohydrate from imported or stored substrates (e.g. sucrose) into the major biosynthetic (e.g. cell wall biosynthesis) and respiratory pathways (e.g. glycolysis and the oxidative pentose phosphate pathway). Disturbing these pathways is expected to have dramatic consequences for germinating pollen grains, which have high metabolic and biosynthetic activities. We propose that residual cPGM mRNA or protein derived from the diploid mother plant is sufficient to enable double mutant female gametophytes to attain maturity and for some to be fertilized. Mature plants possessing a single cPGM allele had a major reduction in cPGM activity. However, photosynthetic metabolism and growth were normal, suggesting that under standard laboratory conditions cPGM activity provided from one wild-type allele is sufficient to mediate the photosynthetic and respiratory fluxes in leaves.

Pentacoordinated phosphorus revisited by high-level QM/MM calculations.

Enzymes catalyzing phosphoryl transfer reactions are extremely efficient and are involved in crucial biochemical processes. The mechanisms of these enzymes are complex due to the diversity of substrates that are involved. The reaction can proceed through a pentacoordinated phosphorus species that is either a stable intermediate or a transition state (TS). Because of this, the first X-ray structure of a pentacoordinated phosphorus intermediate in the beta-phosphoglucomutase enzyme aroused great interest but also much controversy. To provide new insights into the nature of that structure, we have determined the reaction path of the phosphorylation step using high-level QM/MM calculations, and have also calculated the geometry of a complex with a transition state analogue (TSA) that has been suggested to be the actual species in the crystal. The protein crystalline environment has been modeled so as to mimic the experimental conditions. We conclude that the pentacoordinated phosphorus formed in this enzyme is not a stable species but a TS, which gives an activation energy for phosphorylation in agreement with kinetic results. We also show that the TSA is a good mimic of the true TS. We have performed a new crystallographic refinement of the original diffraction map of the pentacoordinated phosphorus structure with the MgF(3)(-) TSA. The new fit improves significantly with respect to the original one, which strongly supports that Allen and coworkers wrongly assigned the X-ray structure to a pentavalent phosphorane.

Glucose 1-phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules.

Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [(14)C]glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

Differences in regulation of carbohydrate metabolism during early fruit development between domesticated tomato and two wild relatives.

Early development and growth of fruit in the domesticated tomato Solanum lycopersicum cultivar Money Maker and two of its wild relatives, S. peruvianum LA0385 and S. habrochaites LA1777, were studied. Although small differences exist, the processes involved and the sequence of events in fruit development are similar in all three species. The growth of developing fruits is exponential and the relative growth rate accelerates from 5 days after pollination (DAP 5) to DAP 8, followed by a decline during further development. Growth is positively correlated to the standard "Brix plus starch'' in the period DAP 8-DAP 20. Carbohydrate composition and levels of sugars and organic acids differ in fruits of the wild accessions compared to domesticated tomato. The wild accessions accumulate sucrose instead of glucose and fructose, and ripe fruits contain higher levels of malate and citrate. The enzymes responsible for the accumulation of glucose and fructose in domesticated tomatoes are soluble invertase and sucrose synthase. The regulation of initial carbohydrate metabolism in the domesticated tomato differs from that in the wild species, as could be concluded from measuring activities of enzymes involved in primary carbohydrate metabolism. Furthermore, changes in the activity of several enzymes, e.g., cell wall invertase, soluble invertase, fructokinase and phosphoglucomutase, could be attributed to changes in gene expression level. For other enzymes, additional control mechanisms play a role in the developing tomato fruits. Localization by in-situ activity staining of enzymes showed comparable results for fruits of domesticated tomato and the wild accessions. However, in the pericarp of S. peruvianum, less activity staining of phosphogluco-isomerase, phosphoglucomutase and UDP-glucosepyrophosphorylase was observed.

Identification of human calphoglin-induced phosphoglucomutase phosphorylation in Escherichia coli.

Orthologous proteomes, universal protein networks conserved from bacteria to mammals, dictate the core functions of cells. To isolate mammalian protein sequences that interact with bacterial signaling proteins, a BLASTP genome search was performed using catalytic domains of bacterial phosphoryl-transfer enzymes as probes. A [32P]phosphoryl-transfer assay of these mammalian cDNA-expressing Escherichia coli cells was used to screen proteins retrieved from the database. Here we report that the expression of a human protein, named calphoglin, resulted in a significant increase in the phosphorylation of a 55-kDa protein in E. coli. The phosphorylation of the 55-kDa protein was acid-stable and its isoelectric point was determined to be 5.4. The 55-kDa protein was sequentially purified from an E. coli extract using three chromatography and two-dimensional polyacrylamide gel electrophoresis. Finally, the 55-kDa protein was purified 830-fold to homogeneity and the N-terminal amino acid sequence was analyzed. The sequence obtained, AIHNRAGQPAQQ, was identical to the N-terminal amino acids of E. coli phosphoglucomutase (PGM). This method may be applicable to the detection and analysis of other orthologous proteomes.

Expression of an Escherichia coli phosphoglucomutase in potato (Solanum tuberosum L.) results in minor changes in tuber metabolism and a considerable delay in tuber sprouting.

The aim of this work was to evaluate the influence of elevating the cytosolic activity of phosphoglucomutase (PGM; EC 5.4.2.2) on photosynthesis, growth and heterotrophic metabolism. Here we describe the generation of novel transgenic plants expressing an Escherichia coli phosphoglucomutase (EcPGM) under the control of the 35S promoter. These lines were characterised by an accumulation of leaf sucrose, despite displaying no alterations in photosynthetic carbon partitioning, and a reduced tuber starch content. Determinations of the levels of a wide range of other metabolites revealed dramatic reductions in maltose and other sugars in leaves of the transformants, as well as a modification of the pattern of organic and amino acid content in tubers of these lines. Intriguingly, the transgenics also displayed a dramatically delayed rate of sprouting and significantly enhanced rate of respiration, however, it is important to note that the severity of these traits did not always correlate with the level of transgene expression. These results are discussed in the context of current understanding of the control of respiration and the breaking of tuber dormancy.

Tuber-specific cytosolic expression of a bacterial phosphoglucomutase in potato (Solanum tuberosum L.) dramatically alters carbon partitioning.

Constitutive antisense inhibition of the cytosolic isoform of phosphoglucomutase in the potato (Solanum tuberosum L.) results in restriction of photosynthesis, growth inhibition and modified tuber morphology, and a severe restriction of tuber starch synthesis. Here we describe the consequences of the tuber-specific expression of an Escherichia coli phosphoglucomutase in the cytosol. Analysis of [14C]glucose metabolism by tuber discs isolated from wild type and transformants revealed that the rates of sucrose and starch synthesis were unaltered but that the rate of glycolysis was depressed in the transgenics. The transformant tubers also contained dramatically reduced amino acid content and significantly higher levels of ADP, but were characterized by elevated levels of Krebs cycle intermediates and an unaltered rate of respiration. In addition to these metabolic consequences of the overexpression of the E. coli enzyme, we observed morphological changes in tubers, with the transformants having a smaller number of larger tubers which exhibited delayed rates of sprouting with respect to the wild type. These results are discussed with respect to current models of the regulation of central plant metabolism and tuber dormancy.

Phosphoryl transfer enzymes and hypervalent phosphorus chemistry.

The coordination tendencies of phosphorus to form a hexacoordinated state from a pentacoordinated state, which might assist in describing the mechanistic action of phosphoryl transfer enzymes, are delineated. The factors discussed include substrate and transition or intermediate state anionicity, hydrogen bonding, packing effects, that is, van der Waals forces, the ease of formation of hexacoordinate phosphorus from lower coordinate states, and the pseudorotation problem common to nonrigid pentacoordinate phosphorus. In view of the work reported in this Account and recent work on enzyme promiscuity and moonlighting activities, it is suggested that donor action should play a role in determining active site interactions in phosphoryl transfer enzyme mechanisms.

The pentacovalent phosphorus intermediate of a phosphoryl transfer reaction.

Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated beta-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate.

The influence of cytosolic phosphoglucomutase on photosynthetic carbohydrate metabolism.

The aim of this work was to examine the role of cytosolic phosphoglucomutase (cPGM; EC 5.4.2.2) in photosynthetic carbon partitioning. We have previously described the generation and characterisation of the tuber metabolism of transgenic potato ( Solanum tuberosum cv. Desiree) lines expressing the StcPGM gene in the antisense orientation under the control of the 35S promoter. Here we extend the characterisation of leaf metabolism within these lines, examining properties of gas exchange, carbon partitioning, and the effect of the genetic manipulation on a wide range of metabolites including metabolites of the sucrose-starch transition, glycolysis, the Krebs cycle and amino acid metabolism. The data acquired in the present study surprisingly reveal that the photosynthetic sucrose synthetic capacity of the leaves is largely unaltered but that these plants display a reduced rate of photosynthesis, a dramatic reduction in nucleotide levels, and a general decline of biosynthesis. We conclude that these lines exhibit only moderate changes in sucrose synthesis but more complex changes on a range of diverse metabolic pathways.

Carbon assimilation and metabolism in potato leaves deficient in plastidial phosphoglucomutase.

We have previously described the generation of transgenic potato ( Solanum tuberosum L. cv. Desiree) lines expressing the S. tuberosum plastidial phosphoglucomutase ( StpPGM) gene in the antisense orientation under the control of the 35S promoter and characterised heterotrophic metabolism in these lines [E. Tauberger et al. (2000) Plant J 23:43-53]. The aim of the current work was to examine the role of plastidial phosphoglucomutase (pPGM, EC 5.4.2.2) in photosynthetic carbon partitioning. Here we characterise the metabolism of leaves of the same lines and show that reducing the activity of this enzyme has profound effects on carbon partitioning, characterised by a strong (up to 50%) reduction in the rate of starch accumulation accompanied by a minor reduction in the rate of sucrose accumulation. Gas-exchange and (14)CO(2)-feeding experiments revealed that the transgenic lines exhibited a decreased rate of photosynthesis and a corresponding reduced assimilation of radiolabel into starch, even in lines exhibiting only a minor decrease in pPGM activity. In illuminated leaves, decreasing the amount of pPGM resulted in decreased amounts of triose-phosphates, hexose-phosphates and inorganic phosphate without changes in the level of 3-phosphoglycerate. Most importantly, the deduced ratio of phosphoesters to inorganic phosphate increased, indicating the likelihood that photosynthesis was phosphate-limited in these lines. Determination of a more complete metabolic profile of leaf material from these lines revealed a large number of changes in the levels of amino and organic acids, consistent with an inhibition of triose-phosphate export from the chloroplast, but little change in the energy status of the transformants. We discuss the implications of these changes with respect to both consequences of inhibiting starch synthesis and of inhibiting photosynthesis, and conclude that a high activity of pPGM is required both to prevent phosphate limitation of photosynthesis and for co-ordination of plastidially and cytosolically compartmented photosynthetic metabolism.

Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism.

The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.

In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues.

A powerful technique is described to localize the activities of a range of enzymes in a wide variety of plant tissues. The method is based on the coupling of the enzymatic reaction to the reduction of NAD and subsequent reduction and precipitation of nitroblue tetrazolium. Enzymes that did not reduce NAD could be visualized by coupling their activities to glucose-6-phosphate dehydrogenase activity via one or more intermediary 'coupling' enzymes. The method is shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase, uridine 5'-diphospho-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be used for all tissues tested, including green leaves, stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution, giving information at the cellular and the subcellular level. The localization of sucrose synthase, invertase, and uridine 5'-diphospho-glucose pyrophosphorylase in transgenic potato plants, carrying a cytokinin biosynthesis gene, is studied and compared with wild-type plants.

The contribution of plastidial phosphoglucomutase to the control of starch synthesis within the potato tuber.

The aim of this work was to evaluate the extent to which plastidial phosphoglucomutase (PGM) activity controls starch synthesis within potato (Solanum tuberosum L. cv. Desirée) tubers. The reduction in the activity of plastidial PGM led to both a correlative reduction in starch accumulation and an increased sucrose accumulation. The control coefficient of plastidial PGM on the accumulation of starch was estimated to approximate 0.24. The fluxes of carbohydrate metabolism were measured by investigating the metabolism of [U-14C]glucose in tuber discs from wild-type and transgenic plants. In tuber discs the control coefficient of plastidial PGM over starch synthesis was estimated as 0.36, indicating that this enzyme exerts considerable control over starch synthesis within the potato tuber.

The plastidic phosphoglucomutase from Arabidopsis. A reversible enzyme reaction with an important role in metabolic control.

An Arabidopsis cDNA (AtPGMp) encoding the plastidic phosphoglucomutase (PGM) predicted a 623-amino acid protein with an N-terminal sequence typical of a plastid signal peptide. Expression of a recombinant protein in Escherichia coli confirmed its enzyme activity. The recombinant enzyme had an apparent K(m) value of 98.5 microM and a V(max) of 4.48 micromol min(-1) (mg protein)(-1). The Calvin cycle intermediates fructose-1,6-bisphosphate and ribulose-1, 5-bisphosphate exerted an inhibitory effect on PGM activity, supporting its proposed involvement in controlling photosynthetic carbon flow. A point mutation was identified in the AtPGMp gene of the Arabidopsis pgm-1 mutant. The mutation in the mutant transcript generated a stop codon at about one third of the wild-type open reading frame, and thus rendered the polypeptide nonfunctional. Storage lipid analysis of the pgm-1 mutant seeds showed a 40% reduction in oil content compared with that of wild type. Our results indicate that plastidic PGM is an important factor affecting carbon flux in triacylglycerol accumulation in oilseed plants, most likely through its essential role in starch synthesis.