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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 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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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