Duplicated gene clusters suggest an interplay of glycogen and trehalose metabolism during sequential stages of aerial mycelium development in Streptomyces coelicolor A3(2)

DNA sequencing and operon disruption experiments indicate that the genes glgBI and glgBII, which code for the two developmentally specific glycogen branching enzymes of Streptomyces coelicolor A3(2) each form part of larger duplicated operons consisting of at least four genes in the order pep1-treS-pep2-glgB. The sequences of the TreS proteins are 73% identical (93% similar) to that of an enzyme that converts maltose into trehalose in Pimelobacter, a distantly related actinomycete; and the Pep1 proteins show relatedness to the α-amylase superfamily. Disruptions of each operon have spatially specific effects on the nature of glycogen deposits, as assessed by electron microscopy. Upstream of the glgBI operon, and diverging from it, is a gene (glgP) that encodes a protein resembling glycogen phosphorylase from Thermatoga maritima and a homologue in Mycobacterium tuberculosis. These three proteins form a distinctive subgroup compared with glycogen phosphorylases from most other bacteria, which more closely resemble the enzymes from eukaryotes. Diverging from the glgBII operon, and separated from the pep1 gene by two very small ORFs, is a gene (glgX) encoding a probable glycogen debranching enzyme. It is suggested that most of these gene products participate in the developmentally modulated interconversion of immobile, inert glycogen reservoirs, and diffusible forms of carbon, both metabolically active (e.g. glucose-1-phosphate generated by glycogen phosphorylase) and metabolically inert but physiologically significant (trehalose). Received: 12 November 1999 / Accepted: 31 January 2000     

Glycogen debranching enzyme in bovine brain

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.     

Comparative Analysis of the glg Operons of Pectobacterium chrysanthemi PY35 and Other Prokaryotes

A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Omega mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. gamma-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.     

Enzymes of glycogen mobilization in the photosynthetic procaryote,Anacystis nidulans

Glycogen, the principal storage compound of assimilatory products in Anacystis nidulans, is synthesized in the light and degraded in the dark. ¹⁴C-labelled glycogen and its radioactive limit dextrin obtained by phosphorylase action were used as substrates to identify enzymes involved in glycogen mobilization. A crude homogenate of cells kept in the dark contained the following enzymes: glycogen phosphorylase (EC 2.4.1.1.) that is firmly bound to glycogen, a debranching enzyme that hydrolyzes 1,6--glucosidic bonds, and an -glucosidase (EC 3.2.1.20). Other amylolytic enzymes were not detectable Using ion exchange chromatography on DEAE-cellulose, -glucosidase and the debranching enzyme could be partly separated from each other and completely from the phosphorylase-glycogen complex. On the basis of their known substrate specificities, the cooperation of these 3 enzymes is sufficient to account for the complete conversion of glycogen into glucose and glucose 1-phosphate.     

Structural rationale for the short branched substrate specificity of the glycogen debranching enzyme GlgX

Glycogen serves as major energy storage in most living organisms. GlgX, with its gene in the glycogen degradation operon, functions in glycogen catabolism by selectively catalyzing the debranching of polysaccharide outer chains in bacterial glycosynthesis. GlgX hydrolyzes α‐1,6‐glycosidic linkages of phosphorylase‐limit dextrin containing only three or four glucose subunits produced by glycogen phosphorylase. To understand its mechanism and unique substrate specificity toward short branched α‐polyglucans, we determined the structure of GlgX from Escherichia Coli K12 at 2.25 Å resolution. The structure reveals a monomer consisting of three major domains with high structural similarity to the subunit of TreX, the oligomeric bifunctional glycogen debranching enzyme (GDE) from Sulfolobus. In the overlapping substrate binding groove, conserved residues Leu270, Asp271, and Pro208 block the cleft, yielding a shorter narrow GlgX cleft compared to that of TreX. Residues 207–213 form a unique helical conformation that is observed in both GlgX and TreX, possibly distinguishing GDEs from isoamylases and pullulanases. The structural feature observed at the substrate binding groove provides a molecular explanation for the unique substrate specificity of GlgX for G4 phosphorylase‐limit dextrin and the discriminative activity of TreX and GlgX toward substrates of varying lengths. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (M_r) of 102069 Da, similar in size to its M_r of about 100 000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3′-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.     

Synthesis of Oleanolic Acid Dimers as Inhibitors of Glycogen Phosphorylase

Recently, oleanolic acid was found to be an inhibitor of glycogen phosphorylase. For further structural modification, we have synthesized several dimers of oleanolic acid by using amide, ester, or triazole linkage with click chemistry. The click chemistry was shown to be the most efficient method for the dimer synthesis. Nearly quantitative yield of triazole‐linked dimers was obtained. Biological evaluation of the synthesized dimers as inhibitors of glycogen phosphorylase has been described. Four of six dimers exhibited inhibitory activity against rabbit muscle glycogen phosphorylase a (RMGPa), with compounds 2 and 7 as the most potent inhibitors, which displayed an IC₅₀ value (ca. 3 μM ) lower than that of oleanolic acid (IC₅₀=14 μM ).     

Role of the Escherichia coli glgX gene in glycogen metabolism

A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.     

Comparison of enzyme activities on glycogen metabolism in rabbit slow and fast muscles

Activities of glycogen synthase (total) and branching enzyme in slow (soleus) muscle are higher than those in fast (vastus lateralis) muscle, while those of phosphorylase kinase (total), phosphorylase (total) and debranching enzyme are reversed. The active form ratio of glycogen synthase is higher in fast muscle, while those of phosphorylase kinase and phosphorylase are higher in slow muscle. Activities of cAMP-dependent protein kinase and protein phosphatase in slow muscle are higher than those in fast muscle. These results suggest that glycogen metabolizing enzymes in slow muscle, distinct from those in fast muscle, are regulated more strongly by cAMP-dependent protein kinase rather than by protein phosphatase.     

Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.     

Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.     

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.     

Effect of Diabetes on Glycogen Metabolism in Rat Retina

Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Molecular Basis of Impaired Glycogen Metabolism during Ischemic Stroke and Hypoxia

Background

Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain''s ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke.

Results

Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia.

Conclusion

Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity.     

Glycogen hyperaccumulation in Saccharomyces cerevisiae ras2 mutant. A biochemical study.

The mechanism by which yeast ras2 mutant hyperaccumulates glycogen has been investigated. Total glycogen synthase activity was between and 1.3 times higher in the ras2 mutant than in an isogenic strain. In addition, while in the normal strain the glycogen synthase activation state decreased along the exponential phase, in the mutant strain the opposite behaviour was observed: glycogen synthase activation state rose continuously reaching full activation at the beginning of the stationary phase. Glycogen phosphorylase a activity was up to 40 times higher in the mutant than in the normal strain. Glucose 6-phosphate and fructose 2,6-bisphosphate levels were slightly more elevated in the mutants. The increase in total glycogen synthase and, particularly, the full activation of this enzyme may explain glycogen hyperaccumulation in the ras2 mutant even in the presence of elevated levels of glycogen phosphorylase a.     

Genetic heterogeneity and the diagnosis of hepatic glycogenoses

Glycogen storage diseases constitute a highly heterogeneous group of disorders, because of the many complex enzyme systems involved in glycogen metabolism, and also because of the diversity of molecular defects connected with gene mutations. To illustrate these features, the authors studied four types of liver glycogen storage diseases, respectively caused by deficiencies of glucose-6-phosphatase, debranching enzyme, phosphorylase and phosphorylase kinase. In each case, the role and functional characteristics of the enzyme system are described, as well as the bioclinical aspects of the deficiency. The only reliable way of diagnosing glycogen storage disease is by assaying the activity of the enzyme concerned. Assay procedure must take account of various factors, especially the progress made in understanding the nature and mechanism of action of enzyme systems, the possible tissular heterogeneity of the deficiency and the functional characteristics of certain enzymes.     

The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp,Crangon crangon

Summary The mobilization of glycogen and phosphoarginine during work and their resynthesis during periods of recovery were investigated in abdominal muscles of the shrimpCrangon crangon. All parameters, metabolite levels as well as glycogen phosphorylase (EC 2.4.1.1) and synthase (EC 2.4.1.11) activities were determined in each individual shrimp investigated. At the onset of work both glycogen and phosphoarginine were degraded with the rate of phosphoarginine utilization being more than 80-fold faster than glycogen. After exhaustive work phosphoarginine stores were replenished within 30 min and seemed to exceed the resting level thereafter. In contrast, glycogen was not resynthesized immediately after work, but was further degraded during recovery leading to the accumulation of lactate. Only when the phosphagen level had reached the resting level did glycogenolysis shift to its resynthesis. The shift is characterized by: (1) a change in the mass action ratio of phosphoglucomutase from values below the equilibrium constant to values above the constant, (2) a dramatic decrease in the ratio fructose 1,6-bisphosphate/fructose 6-phosphate indicating phosphofructokinase inhibition, (3) an increase in the glucose concentration, and (4) an increase in the proportion of glycogen synthase I. The inactivation of glycogen phosphorylase by dephosphorylation during recovery was 2.4-fold. 36±8% (n=5) of total activity remained in the phosphorylated form. It is proposed that this part of the enzyme was inactivated by the drop in inorganic phosphate level due to the restoration of phosphoarginine.     

Actions of glycogen synthase and prosphorylase of rabbit-skeletal muscle on modified glycogens

The high reactivities exhibited by rabbit-muscle synthase and phosphorylase for unmodified glycogen-acceptors decrease progressively, presumably because of a large increase in apparent K_m as the glycogen molecule is converted into its component maltosaccharide chains by the debranching enzyme, isoamylase. Elongation of the outer chains of glycogen acceptor also results in decreased reactivities of the two transglucosylases and this is shown, for phosphorylase acting in the direction of glucan synthesis, to be caused by a decrease in the V_max of the reaction. A partial restoration of the degradative reactivity of phosphorylase by a limited alpha-amylolysis of the long outer-chains of modified glycogen suggests a role of cytoplasmic alpha-amylase in mammalian glycogen metabolism.     

Synthesis of a C-glucosylated cyclopropylamide and evaluation as a glycogen phosphorylase inhibitor

The synthesis of carbohydrate-based glycogen phosphorylase inhibitors is attractive for potential applications in the treatment of type 2 diabetes. A titanium-mediated synthesis led to a benzoylated C-glucosylated cyclopropylamine intermediate, which underwent a benzoyl migration to afford the corresponding 2-hydroxy-C-glycoside. X-ray crystallographic studies revealed a unit cell composed of four molecules as pairs of dimers connected through two hydrogen bonds. The deprotection of the benzoate esters under Zemplén conditions afforded a glycogen phosphorylase inhibitor candidate displaying weak inhibition toward glycogen phosphorylase (16% at 2.5 mM).