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Journal of Bacteriology, July 2007, p. 5325-5333, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00481-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Regions Critically Affecting Kinetics and Allosteric Regulation of the Escherichia coli ADP-Glucose Pyrophosphorylase by Modeling and Pentapeptide-Scanning Mutagenesis{triangledown}

Miguel A. Ballicora,1* Esteban D. Erben,2,{dagger} Terutaka Yazaki,3 Ana L. Bertolo,3,{ddagger} Ana M. Demonte,2 Jennifer R. Schmidt,3 Mabel Aleanzi,2 Clarisa M. Bejar,3 Carlos M. Figueroa,2 Corina M. Fusari,2,§ Alberto A. Iglesias,2 and Jack Preiss3

Department of Chemistry, Loyola University, Chicago, Illinois 60626,1 Laboratorio de Enzimología Molecular, Cátedra de Bioquímica Básica de Macromoléculas, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina,2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 488243

Received 29 March 2007/ Accepted 30 April 2007

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.

* Corresponding author. Mailing address for Miguel A. Ballicora: Department of Chemistry, Loyola University, Chicago, IL 60626. Phone: (773) 508-3154. Fax: (773) 508-3086. E-mail: mballic{at}luc.edu. Mailing address for Jack Preiss: Dept. of Biochemistry & Molecular Biology, Michigan State University, E. Lansing, MI 48824. Phone: (517)353-3137. Fax: (517)353-9334. E-mail: preiss{at}msu.edu

{triangledown} Published ahead of print on 11 May 2007.

{dagger} Present address: INGEBI, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina.

{ddagger} Present address: Departamento de Genética, Escola Superior de Agricultura "Luiz de Queiroz," Universidade de São Paulo, Av. Pádua Dias 11, CP 83, CEP 13400-970, Piracicaba, SP, Brazil.

§ Present address: INTA, Castelar, Pcia. de Buenos Aires, Argentina.

Journal of Bacteriology, July 2007, p. 5325-5333, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00481-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Georgelis, N., Shaw, J. R., Hannah, L. C. (2009). Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme. Plant Physiol. 151: 67-77 [Abstract] [Full Text]  


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