An unregulated UDP-glucose pyrophosphorylase that acquires allosteric regulation by domain addition

ASENCIÓN DIEZ, MATÍAS D.; EBRECHT, ANA C.; MARTINEZ, LUCILA I.; ALEANZI, MABEL C.; GUERRERO, SERGIO A.; BALLÍCORA, MIGUEL A.; IGLESIAS, ALBERTO A.

Chicago, Illinois

Conferencia; The 33rd Midwest Enzyme Chemistry Conference (MECC); 2013

In bacteria, sugar anabolism involves partition of glucose-1-phosphate (Glc-1P) into either ADP-Glc or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (ADP-Glc PPase) or unregulated UDP-Glc PPase2,3. Previously, we characterized the Streptococcus mutans UDP-Glc PPase (SmuUDP-Glc PPase) and constructed a chimeric protein by adding the C-terminal domain from the Escherichia coli ADP-Glc PPase to the entire SmuUDP-Glc PPase (SmuGalU-Δ294EcoGlgC)1. Both proteins were fully active as UDP-Glc PPases. After characterization, the chimeric enzyme showed higher affinity for substrates than the native SmuUDP-Glc PPase, but the maximal activity was 4-fold lower. Interestingly, the chimeric protein was sensitive to allosteric regulation by pyruvate, 3-phosphoglycerate 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 3-fold and increased the affinity for substrates. Thus, we highlighted the importance of the C-terminal domain in acquiring sensitivity to allosteric effector in PPases, being this chimeric protein the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, it was suggested an interplay between both the N- and the C-terminal domains in the allosteric response from prokaryotic ADP-Glc PPases2. In this context, we further investigated the regulatory properties of the chimeric enzyme SmuGalU-Δ294EcoGlgC. Then, we constructed the SmuGalU-Δ294EcoGlgC A26K, L27R and A26KL27R mutants. In this way, we added residues belonging to the bacterial ADP-Glc PPase N-terminal domain involved in binding allosteric effectors2. The mutants were characterized regarding kinetic and regulatory properties. Our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.