ADP-glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis

Abstract

The accumulation of alpha-1,4-polyglucans is an important strategy to cope with transient starvation conditions in the environment. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing ADP-glucose as the glucosyl donor for elongation of the alpha-1,4-glucosidic chain. The main regulatory step takes place at the level of ADP-glucose synthesis, a reaction catalyzed by ADP-Glc pyrophosphorylase (PPase). Most of the ADP-Glc PPases are allosterically regulated by intermediates of the major carbon assimilatory pathway in the organism. Based on specificity for activator and inhibitor, classification of ADP-Glc PPases has been expanded into nine distinctive classes. According to predictions of the secondary structure of the ADP-Glc PPases, they seem to have a folding pattern common to other sugar nucleotide pyrophosphorylases. All the ADP-Glc PPases as well as other sugar nucleotide pyrophosphorylases appear to have evolved from a common ancestor, and later, ADP-Glc PPases developed specific regulatory properties, probably by addition of extra domains. Studies of different domains by construction of chimeric ADP-Glc PPases support this hypothesis. In addition to previous chemical modification experiments, the latest random and site-directed mutagenesis experiments with conserved amino acids revealed residues important for catalysis and regulation.

FIG. 1.

Alignment of ADP-Glc PPases from different classes. Amino acid alignment was performed with the program PILEUP from the Wisconsin package (http://www.gcg.com). The alignment was fine tuned manually based on the secondary structure of each enzyme as predicted by the PHD program (81). Residues in blue and red were predicted to be β-sheets and α-helixes, respectively; pale shades indicate a lower level of confidence. Green residues were predicted to be neither of these (loops). In black are residues for which the PHD program could not make a prediction. Insertions and deletions were introduced to maximize the alignment of both primary and secondary structure. a, sequence of the small (catalytic) subunit; b, sequence of the subunit encoded by glgC (catalytic).

FIG. 1.

Alignment of ADP-Glc PPases from different classes. Amino acid alignment was performed with the program PILEUP from the Wisconsin package (http://www.gcg.com). The alignment was fine tuned manually based on the secondary structure of each enzyme as predicted by the PHD program (81). Residues in blue and red were predicted to be β-sheets and α-helixes, respectively; pale shades indicate a lower level of confidence. Green residues were predicted to be neither of these (loops). In black are residues for which the PHD program could not make a prediction. Insertions and deletions were introduced to maximize the alignment of both primary and secondary structure. a, sequence of the small (catalytic) subunit; b, sequence of the subunit encoded by glgC (catalytic).

FIG. 2.

Prediction of secondary structure of ADP-glucose pyrophosphorylases. The secondary structure of various ADP-Glc PPases from bacteria as well as plants was predicted with the PHD program (81). The secondary structures align very well (19) with the sequences of UDP-N-acetylglucosamine pyrophosphorylase (11)and TDP-glucose pyrophosphorylase (8). Sequences shown as arrows are predicted to be β-pleated sheets, and sequences shown as cylinders are predicted to be α-helices. These structures are interconnected with amino acid sequences indicated as being neither α-helices or β-pleated sheets and are possibly random structures or loops. They are shown as lines. White triangles indicate areas where proteinase K hydrolyzes the E. coli enzyme (99). Black triangles indicate where the Anabaena ADP-Glc PPase is partially proteolyzed by trypsin, and gray triangles indicate partial hydrolysis of the E. coli enzyme by trypsin (unpublished data). The proteolysis results suggest that the areas sensitive to proteases are exposed random structures (loops). Residues K³⁹, Y¹¹⁴, and K¹⁹⁵ are the amino acids in the E. coli ADP-Glc PPase that bind the activator fructose 1,6-bisphosphate and the substrates ATP and Glc 1-phosphate, respectively. D¹⁴² is the amino acid shown to be a catalytic residue in the E. coli enzyme. P²⁹⁵ and G³³⁶ are amino acids that, when mutated, affect the allosteric properties of the ADP-Glc PPase (59, 60). Regions 1, 2, and 3 form the putative catalytic domain, and region 4 may also be part of the catalytic domain, as suggested by alignment with the crystal structures of UDP-N-acetylglucosamine pyrophosphorylase (11) and TDP-glucose pyrophosphorylase (8).