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 18.104.22.168, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 22.214.171.124). 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.
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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.
Asención Diez MD, Ebrecht AC, Martínez LI, Aleanzi MC, Guerrero SA, Ballícora MA, Iglesias AA. A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators. International Journal of Molecular Sciences. 2013; 14(5):9703-9721.
Asención Diez, Matías D.; Ebrecht, Ana C.; Martínez, Lucila I.; Aleanzi, Mabel C.; Guerrero, Sergio A.; Ballícora, Miguel A.; Iglesias, Alberto A. 2013. "A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators." Int. J. Mol. Sci. 14, no. 5: 9703-9721.