De novo synthesis of Escherichia coli glycogen is due to primer associated with glycogen synthase and activation by branching enzyme.

Previous reports have demonstrated the incorporation of glucose from ADP-glucose into methanol-insoluble and TCA-insoluble fractions in cell extracts of Escherichia coli in the absence of added primer α-glucan. This activity is reduced 6- to 76-fold in cell extracts of three independently isolated glycogen synthase-deficient mutants of E. coli B. Homogeneous preparations of E. coli B glycogen synthase catalyze incorporation of glucose into both methanol- and TCA-insoluble fractions in the absence of added primer. Since glycogen synthase catalyzes these reactions, it is not necessary to propose a protein acceptor glucose or a unique ADP-glucose-glycosyl transferase to catalyze formation of the glucoprotein in E. coli cell extracts to explain glucose incorporation into TCA-insoluble material (R. Barengo et al. (1975) FEBS Lett.53, 274–278). The incorporation of glucose into methanol-and TCA-insoluble fractions is stimulated by 0.25 m citrate and by branching enzyme. Citrate reduces the K_m for the primer, glycogen, about 11- to 15-fold. Branching enzyme can also reduce the concentration of primer required for incorporation of glucose into methanol-insoluble material. The simultaneous presence of both 0.25 m citrate and branching enzyme enables the glycogen synthase reaction rate to proceed at 30% the maximal velocity at a primer concentration of 1 μg/ml. Incorporation of glucose into methanol- or TCA-insoluble material in the absence of primer is completely inhibited by adding α-amylase. Furthermore, incorporation into methanol- or TCA-insoluble material is reduced 13- to 16-fold relative to the reaction occurring in the presence of primer when glycogen synthase is pretreated with glucoamylase and α-amylase. Previous results show that homogeneous preparations of glycogen synthase contain glucan. Heat-denatured glucogen synthase can act as a primer for the glycogen phosphorylase and glycogen synthase reactions. Both the TCA- and methanol-insoluble products form I₂-glucan complexes with wavelength maxima of about 580–590 nm and 610–615 nm, respectively, suggesting that they are mainly linear chain glucans. The products are completely solubilized with α-amylase. The TCA-insoluble product is not solubilized by pronase treatment. The above results strongly suggest that previous reports on formation of glucoprotein primer for glycogen synthesis or on de novo glycogen synthesis in various similar systems is due to endogenous glucan associated with glycogen synthase rather than formation of glucoprotein which then acts as primer for glycogen synthesis.     

Immunological characterization of Escherichia coli B glycogen synthase and branching enzyme and comparison with enzymes from other bacteria.

Escherichia coli B glycogen synthase and branching enzyme, although similar in amino acid composition, had no significant immunological cross-reactivity. The N-terminal sequences of the glycogen synthase were rich in hydrophobic residues, whereas branching enzyme had a higher content of acidic and basic residues. However, residues 21 to 28 of glycogen synthase and 7 to 14 of branching enzyme shared six of eight residues in common. Two fractions of branching enzyme, branching enzymes I and II, which can be isolated from E. coli B cell extracts, have been shown to be immunologically identical, suggesting that only one type of branching enzyme activity is present in E. coli B. Evidence has been obtained which indicates that E. coli B glycogen synthase and branching enzyme are antigenically very similar to glycogen synthases and branching enzymes from other enteric bacteria. No cross-reactivity with either enzyme was observed in cell extracts from photosynthetic bacteria.     

Characterization and crystallization of an active N-terminally truncated form of the Escherichia coli glycogen branching enzyme.

The prokaryotic glycogen branching enzymes (GBE) can be divided into two groups on the basis of their primary structures: the first group of enzymes, which includes GBE from Escherichia coli, is characterized by a long N-terminal extension that is absent in the enzymes of the second group. The extension consists of approximately 100 amino-acid residues with unknown function. In order to characterize the function of this region, the 728 amino-acid residue, full-length E. coli GBE, and a truncated form (nGBE) missing the first 107 amino-acid residues were overexpressed in E. coli. Both enzymes were purified to homogeneity by a simple purification procedure involving ammonium sulphate precipitation, ion-exchange chromatography, and a second ammonium sulphate precipitation. Purified full-length enzyme was poorly soluble and formed aggregates, which were inactive, at concentrations above 1 mg.mL-1. In contrast, the truncated form could be concentrated to 6 mg.mL-1 without any visible signs of aggregation or loss of activity on concentration. The ability to overexpress nGBE in a highly soluble form has allowed us to produce diffracting crystals of a branching enzyme for the first time. A comparison of the specific activities of purified GBE and nGBE in assays where amylose was used as substrate demonstrated that nGBE retained approximately half of the branching activity of full-length GBE and is therefore a suitable model for the study of the enzymes' catalytic mechanism.     

Expression of Escherichia coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch‐like polyglucans

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water‐insoluble and semi‐crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water insolubility, crystallinity and presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen BE (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water‐soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis, which is devoid of BE activity and consequently free of starch. The synthesis of a water‐insoluble, partly crystalline, amylose‐containing starch‐like polyglucan was restored in GlgB‐expressing plants, suggesting that BEs' origin only has a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water‐soluble, poorly crystalline polyglucan.     

Mode of action of glycogen branching enzyme from Neurospora crassa

Neurospora crassa branching enzyme [EC 2.4.1.18] acted on potato amylopectin or amylose to convert them to highly branched glycogen-type molecules which consisted of unit chains of six glucose units. The enzyme also acted on the amylopectin beta-limit dextrin, indicating that the enzyme acted on internal glucose chains as well as outer chains. By the combined action of N. crassa glycogen synthase [EC 2.4.1.11] and the branching enzyme, a glycogen-type molecule was formed from UDP-glucose. In the presence of primer glycogen, the glucose transfer reaction was accelerated by the addition of branching enzyme. On the other hand, the glucose transfer reaction by glycogen synthase did not occur without primers. When the branching enzyme was added, the glucose transfer occurred after a short time lag. This recovery of the glucose transfer reaction did not occur upon addition of the inactivated branching enzyme. The structure of the product formed by the combined action of the two enzymes was different from that of the intact N. crassa glycogen with respect to the distribution patterns of the unit chains.     

Limited proteolysis of branching enzyme from Escherichia coli

Branching enzyme is involved in determining the structure of starch and glycogen. It catalyzes the formation of branch points by cleavage and transfer of alpha-1,4-glucan chains to alpha-1,6 branch points. Branching enzyme belongs to the amylolytic family of enzymes containing four conserved regions in a central (alpha/beta)8-barrel. Limited proteolysis of the branching enzyme from Escherichia coli (84 kDa) by proteinase K produced a truncated protein of 70-kDa, which still retained 40-60% of branching activity, depending on the type of assay used. Amino acid sequencing showed that the 70-kDa protein lacked 111 or 113 residues at the amino terminal, whereas the carboxy terminal was still intact. We purified this truncated enzyme to homogeneity and analyzed its properties. The enzyme had a three- to fourfold lower catalytic efficiency than the native enzyme, whereas the substrate specificity was unaltered. Furthermore, a branching enzyme with 112 residues deleted at the amino terminal was constructed by recombinant technology and found to have properties identical to those of the proteolyzed enzyme.     

Characterization of Escherichia coli B glycogen synthase enzymatic reactions and products.

Previous reports implicate UDPglucose as an active glucosyl donor for the unprimed reaction and “glucoprotein” formation in glycogen biosynthesis in Escherichia coli. Results presented here indicate that UDPglucose and GDPglucose are glucosyl donors in the primed and unprimed reactions catalyzed by purified E. coli B glycogen synthase at less than 5% the rate observed when ADPglucose is the donor. The unprimed reaction is stimulated by 0.25 m citrate and a high molecular weight product is formed similar to that produced when ADPglucose is the glucosyl donor. Physiological amounts of branching enzyme and high concentrations of glycogen inhibit transfer from UDPglucose and GDPglucose. In addition, transfer from UDPglucose is inhibited by ADPglucose. These results strongly suggest that ADPglucose is the physiological donor in both the primed and unprimed reactions. Furthermore, these and previously reported results suggest that one enzyme is involved in the catalysis of the primed, unprimed, and TCA-insoluble product formation reactions. Antiserum prepared against purified E. coli B glycogen synthase inactivates transfer of glucose from either ADPglucose or UDPglucose in the above reactions catalyzed by E. coli B crude extracts. Purified E. coli B glycogen synthase preparations contain significant amounts of α-glucan primer. Evidence shows that this glucan is not covalently attached to the enzyme. Results presented show that formation of material insoluble in TCA and previously considered to be due to “glucoprotein” formation, is in fact due to the generation of long chain length glucan molecules intrinsically acid insoluble. The data suggest that previous results purported to be de novo synthesis of glycogen are due to glucan associated with the glycogen synthase and not to formation of a “glucoprotein” intermediate which then acts as primer for further oligosaccharide synthesis.     

Expression of branching enzyme I of maize endosperm in Escherichia coli.

The gene encoding for mature branching enzyme (BE) I (BEI) of maize (Zea mays L.) endosperm has been expressed in Escherichia coli using the T7 promoter. The expressed BEI was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation. The recombinant enzyme showed properties very similar to those of BEI purified from developing maize endosperm with respect to branching amylose and amylopectin. This result confirmed our earlier report that maize endosperm BEI had a higher rate of branching amylose and a much lower rate (less than 10% of that of branching amylose) of branching amylopectin. This study also showed a great advantage in purifying BEI from the bacterial expression system rather than from developing maize endosperm. Most important, this study has established the system with which to study the structure-function relationships of the maize BEI using site-directed mutagenesis.     

The ADP-glucose binding site of the Escherichia coli glycogen synthase

Bacterial glycogen/starch synthases are retaining GT-B glycosyltransferases that transfer glucosyl units from ADP-Glc to the non-reducing end of glycogen or starch. We modeled the Escherichia coli glycogen synthase based on the coordinates of the inactive form of the Agrobacterium tumefaciens glycogen synthase and the active form of the maltodextrin phosphorylase, a retaining GT-B glycosyltransferase belonging to a different family. In this model, we identified a set of conserved residues surrounding the sugar nucleotide substrate, and we replaced them with different amino acids by means of site-directed mutagenesis. Kinetic analysis of the mutants revealed the involvement of these residues in ADP-Glc binding. Replacement of Asp21, Asn246 or Tyr355 for Ala decreased the apparent affinity for ADP-Glc 18-, 45-, and 31-fold, respectively. Comparison with other crystallized retaining GT-B glycosyltransferases confirmed the striking similarities among this group of enzymes even though they use different substrates.     

The X-ray crystallographic structure of Escherichia coli branching enzyme

Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-A crystal structure of glycogen branching enzyme from Escherichia coli. The enzyme consists of three major domains, an NH(2)-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site. While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase. Oligosaccharide binding was modeled for branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding. While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50 degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme. A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure. It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.     

Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.     

Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1‐suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.

     

Identification and characterization of a critical region in the glycogen synthase from Escherichia coli

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.     

Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis.

Summary The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an M_r of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - ^H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53° C.     

Role of maltose enzymes in glycogen synthesis by Escherichia coli

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.     

Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme.

The yeast glycogen branching enzyme (EC 2.4.1.18) is shown to be induced in batch culture simultaneously with the onset of intracellular glycogen accumulation. The branching enzyme structural gene (GLC3) has been cloned. Its predicted amino acid sequence is very similar to procaryotic branching enzymes. Northern analysis indicates that GLC3 mRNA abundance increases in late exponential growth phase coincident with glycogen accumulation. Disruption of the branching enzyme structural gene establishes that branching enzyme activity is an absolute requirement for maximal glycogen synthesis.     

Detection of two essential sulfhydryl residues in Escherichia coli B glycogen synthase

Results presented indicate that two distinct essential sulfhydryl residues are present in the Escherichia coli B glycogen synthase. One residue is modified by iodoacetic acid and can be protected by ADP or ADPglucose. The other site can be modified by 5,5′-dithiobis (2-nitrobenzoic acid) and is protected by glycogen. Each reagent appears to be specific for a given site and thus allows the two sites to be distingushed.