Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of theEscherichia coliADP-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.

Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

“…In addition, the ADPGlcPPase from potato tuber was found to be postranslationally regulated by redox modification of specific cysteine residues [42]. It has been proposed that ADP-GlcPPase is the only regulatory enzyme between pyrophosphorylases, in agreement with the protein structure showing that the former is a bigger protein having a characteristic C-term domain [26,43]. Regulatory properties/ mechanisms were never found for any UDP-GlcPPase.…”

Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

“…Saturation curves were performed by assaying the respective enzyme activities at saturating levels of a fixed substrate and different concentrations of the variable substrate. The experimental data were plotted as enzyme activity (U/mg) versus substrate (or effector) concentration (mM), and the kinetic constants were determined by fitting the data to the Hill equation as described elsewhere (2). Fitting was performed with the Levenberg-Marquardt nonlinear least-squares algorithm provided by the computer program Origin7.0.…”

Characterization of the CI Repressor Protein Encoded by the Temperate Lactococcal Phage TP901-1

“…cI mutants in which a 15-base pair linker was inserted randomly into the gene were constructed. Insertion of five additional amino acids in a surface loop with no specific target function, in a connector region, or in a relatively unstructured region is expected to have a minor effect on the function of the protein, whereas insertion within tightly folded regions with secondary structures or functionally important loops should have a greater effect (1,2,9). In this study we tested the ability of the mutated cI genes to produce proteins able to repress transcription from P L in vivo.…”