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Int. J. Mol. Sci. 2013, 14(5), 9703-9721; doi:10.3390/ijms14059703
Article

A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

1,2,†
,
1,2,†
,
1
,
1
,
1
,
2
 and
1,*
1 Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina 2 Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W Sheridan Rd., Chicago, IL 60660, USA These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 1 March 2013 / Revised: 10 April 2013 / Accepted: 18 April 2013 / Published: 6 May 2013
(This article belongs to the Special Issue Molecular Cut and Paste)
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Abstract

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.
Keywords: protein engineering; allosteric regulation; pyrophosphorylases evolution; UDP-glucose; ADP-glucose protein engineering; allosteric regulation; pyrophosphorylases evolution; UDP-glucose; ADP-glucose
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Asención Diez, M.D.; Ebrecht, A.C.; Martínez, L.I.; Aleanzi, M.C.; Guerrero, S.A.; Ballícora, M.A.; Iglesias, A.A. A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators. Int. J. Mol. Sci. 2013, 14, 9703-9721.

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