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

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. Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Ballicora, Miguel A.. University Of Chicago; Estados Unidos de América; Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina