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Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2 degrees overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the alpha-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site.

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.

Bacterial glycogen synthases transfer a glucosyl unit, retaining the anomeric configuration, from ADP-glucose to the non-reducing end of glycogen. We modeled the Escherichia coli glycogen synthase based on three glycosyltransferases with a GT-B fold. Comparison between the model and the structure of the active site of crystallized retaining GT-B glycosyltransferases identified conserved residues with the same topology. To confirm the importance of these residues predicted by the model, we studied them in E. coli glycogen synthase by site-directed mutagenesis. Mutations D137A, R300A, K305A, and H161A decreased the specific activity 8100-, 2600-, 1200-, and 710-fold, respectively. None of these mutations increased the Km for glycogen and only H161A and R300A had a higher Km for ADP-Glc of 11- and 8-fold, respectively. These residues were essential, validating the model that shows a strong similarity between the active site of E. coli glycogen synthase and the other retaining GT-B glycosyltransferases known to date.

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.

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.

Glycogen synthases from Escherichia coli and mammalian muscle differ in many respects including regulation, sugar nucleotide specificity, and primary sequence. To compare the structure of the active sites in these enzymes, the affinity-labeling study of the E. coli enzyme was carried out using adenosine diphosphopyridoxal as the reagent. The E. coli enzyme was inactivated in a time- and dose-dependent manner when incubated with the reagent followed by sodium borohydride reduction. The inactivation was markedly protected by ADP-glucose and ADP, suggesting that the reagent was bound to the substrate-binding site. The stoichiometry of the bound reagent to the enzyme was approximately 1:1. Sequence analysis of the labeled peptide isolated from a proteolytic digest of the modified protein revealed that Lys15 is labeled. Based on the geometry of the reagent, the epsilon-amino group of this residue might be located close to the pyrophosphate moiety of ADP-glucose bound to the E. coli enzyme, like that of Lys38 in the rabbit muscle enzyme, which is labeled by uridine diphosphopyridoxal (Tagaya, M., Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 260, 6670-6676; Mahrenholz, A. M., Wang, Y., and Roach, P. J. (1988) J. Biol. Chem. 263, 10561-10567). The importance of the conserved sequence of Lys-X-Gly-Gly is discussed in connection with the glycine-rich region found in many nucleotide-binding proteins.

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.

Structural gene mutants of the glycogen biosynthetic enzymes adenosine diphosphate glucose pyrophosphorylase (glgC) and glycogen synthase (glgA) were isolated and partially characterized. The cotransduction frequencies of these genes with the aspartic semialdehyde dehydrogenase (asd) and glycerol-3-phosphate dehydrogenase (glpD) genes suggested the unambiguous gene order of glpD glgA glgC asd. The results of the three-factor cross glpD- glgA- glgC+ X glpD+ glgA+ glgC- were consistent with the proposed order. A simultaneous and approximately equivalent derepression of the glgC, glgA, and glgB (branching enzyme) gene products was observed in the late logarithmic-early stationary phase of growth on enriched media. These results are consistent with the coordinately regulated synthesis of the three glycogen biosynthetic enzymes in Salmonella typhimurium.

The nucleotide sequence of the glgA gene, coding for glycogen synthase (EC 2.4.1.21) was elucidated. It consists of 1431 base pairs specifying a protein of 477 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis obtained with the pure protein as well as with the molecular weight as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal acid sequence and amino acid sequence analysis of various peptides obtained from CNBr degradation of purified glycogen synthase.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.