Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis

Abstract

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu(102) and Pro(103) was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.

FIG. 1.

Homology models of E. coli ADP-Glc PPase. Models were built as described in Materials and Methods. (A) The model was built on the known structure of dTDP-Glc PPase representing the N-terminal (catalytic) domain of the E. coli ADP-Glc PPase. Residues R32, K42, Y114, D142, and K195 are depicted as balls and sticks, and the protein structure is in a Richardson-style ribbon diagram. Yellow spheres represent pentapeptide insertions that disrupted the enzyme activity. Dark spheres are insertions that did not abolish the activity and stained with iodine. An orange sphere depicts an insertion that eliminated the activation by FBP. Modeled Glc1P is in red and deoxyribose triphosphate in blue. The oversized blue sphere in the deoxyribose triphosphate molecule represents where the adenine moiety would be covalently bound. The purple dot represents a modeled Mg²⁺. (B) Selected elements of the structure in panel A are shown at a different angle. (C) Stereo diagram of the model with the N and C domains of the E. coli ADP-Glc PPase was based on the S subunit from the potato tuber enzyme. The protein structure is in a Richardson-style ribbon diagram, and the substrate ATP is in a space-filling representation. Yellow spheres represent insertions in the C domain that abolished the activity. White spheres are insertions that introduced stop codons. The purple structure is expressed as a separate polypeptide initiated in Met³²⁸ when a stop codon was introduced with insertion 117. The orange sphere represents insertion 8 that abolishes the effect of activator FBP. The orange loop is between residues Gln¹⁰⁵ and Gly¹¹⁶; the Gln¹⁰⁵-Gly¹¹⁶ sequence interacts with the adenine moiety of the ATP and is just after insertion 8. Side chains and C-α depicted are from residues that when subjected to mutagenesis altered the allosteric properties of either the enzyme from E. coli or A. tumefaciens.

FIG. 2.

Insertions in the E. coli ADP-Glc PPase gene. White triangles represent insertions that lead to transformed colonies that did not stain with iodine. Black triangles represent insertions in colonies that synthesized glycogen as detected by brown or black staining of the cells. The black line indicates a loop Gln¹⁰⁵-Gly¹¹⁶ that is predicted to interact with ATP. The black circle indicates Tyr¹¹⁴, which was shown to be close to the ATP site. ins19 was not included in the figure, because it is equivalent to ins4; they had the same sequence.

FIG. 3.

Effect of FBP on the activity of different insertion mutants in crude extracts. Insertion mutants were expressed, and crude extracts were obtained as described in Materials and Methods. Aliquots were assayed in the pyrophosphorolysis direction as described in Materials and Methods in the presence of 1.5 mM FBP (white bars) or in the absence of activator (black bars). WT is the wild-type enzyme expressed with pETEC, and the control is pET24a without insert.

FIG. 4.

Effect of FBP on the apparent affinity for ATP-Mg. Enzyme assays in the synthesis direction of the wild-type enzyme (WT) and Ins8 enzyme were performed as described in Materials and Methods. The concentration of MgCl₂ was 7 mM in the absence of ATP. Variations of ATP were accompanied by equimolar additions of MgCl₂ to ensure saturating concentration of free Mg²⁺ (7 mM). The concentration of FBP was 1.5 mM (white circles), whereas the control reaction mixtures (black triangles) did not contain FBP.