Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of the Escherichia coli ADP-Glucose Pyrophosphorylase by Modeling and Pentapeptide-Scanning Mutagenesis
| Autor: | Esteban Erben, Ana L. Bertolo, Corina M. Fusari, Carlos Maria Figueroa, Alberto A. Iglesias, Jennifer R. Schmidt, Jack Preiss, Terutaka Yazaki, Mabel Cristina Aleanzi, Clarisa M. Bejar, Miguel A. Ballicora, Ana María Magdalena Demonte |
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| Rok vydání: | 2007 |
| Předmět: |
Molecular Sequence Data
Allosteric regulation Magnesium Chloride Glucose-1-Phosphate Adenylyltransferase Biology medicine.disease_cause Microbiology Pentapeptide repeat Catalysis Protein Structure Secondary Substrate Specificity Ciencias Biológicas Structure-Activity Relationship Adenosine Triphosphate Escherichia coli medicine Coding region Amino Acid Sequence Glycogen synthase Molecular Biology chemistry.chemical_classification Sequence Homology Amino Acid Escherichia coli Proteins ADP-GLUCOSE PYROPHOSPHORYLASE Bioquímica y Biología Molecular Enzymes and Proteins Molecular biology Protein Structure Tertiary Amino acid Kinetics Mutagenesis Insertional Enzyme chemistry Biochemistry Genes Bacterial Structural Homology Protein Enzyme model Codon Terminator biology.protein Oligopeptides CIENCIAS NATURALES Y EXACTAS |
| Zdroj: | Journal of Bacteriology. 189:5325-5333 |
| ISSN: | 1098-5530 0021-9193 |
| Popis: | 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 Leu102 and Pro103 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. Fil: Ballicora, Miguel A.. Loyola University; Estados Unidos Fil: Erben, Esteban Daniel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Yazaki, Terutaka. Michigan State University; Estados Unidos Fil: Bertolo, Ana L.. Michigan State University; Estados Unidos Fil: Demonte, Ana María Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina Fil: Schmidt, Jennifer R.. Michigan State University; Estados Unidos Fil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina Fil: Bejar, Clarisa M.. Michigan State University; Estados Unidos Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina Fil: Fusari, Corina M.. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina Fil: Preiss, Jack. Michigan State University; Estados Unidos |
| Databáze: | OpenAIRE |
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