title: A Chimeric UDP-glucose pyrophosphorylase produced by protein engineering exhibits sensitivity to allosteric regulators.
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.
authors: Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina. iglesias@fbcb.unl.edu.ar.
authors: Matías D Asención
authors: MD
authors: Diez
authors: Ana C
authors: AC
authors: Ebrecht
authors: Lucila I
authors: LI
authors: Martínez
authors: Mabel C
authors: MC
authors: Aleanzi
authors: Sergio A
authors: SA
authors: Guerrero
authors: Miguel A
authors: MA
authors: Ballícora
authors: Alberto A
authors: AA
authors: Iglesias
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005958:Glucosephosphates / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013295:Streptococcus mutans / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005958:Glucosephosphates; D011993:Recombinant Fusion Proteins; C031590:glucose-1-phosphate; D051599:Glucose-1-Phosphate Adenylyltransferase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase
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