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.

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis

“…It is worth mentioning that both PEP and Glc-6P were reported as activators of the ADP-Glc PPase from M. smegmatis [58] and S. coelicolor [27] , thus suggesting a common activation in these phylogenetically related actinobacteria. Conversely, Mtb UDP-Glc PPase and GSase were insensitive to the metabolites tested, in agreement with the lack of regulatory properties already reported for both enzymes from prokaryotes [21,23,24,27,44,61] , including that from M. smegmatis [58] .…”

“…Some of these activated sugars are used to build the glycosidic structure of the bacterial cell wall and capsule or more complex oligo and polysaccharides [22,23] . UDP-Glc PPases from prokaryotes are not known to be allosterically regulated [24] , sharing less than 10% identity with their eukaryotic counterparts [21] .…”

A Novel Dual Allosteric Activation Mechanism of Escherichia coli ADP-Glucose Pyrophosphorylase: The Role of Pyruvate

“…On the other hand, those residues were not critical for Pyr activation [7] , [32] . If we accept previous evidence that both domains interact with Fru-1,6-P 2 [11] , [33] and Pyr mainly with the C-terminus [5] , [34] , we can postulate the activation scheme in Figure 6 for the E. coli ADP-Glc PPase. In that scheme, Pyr binds to the C-domain and as a consequence, it activates the enzyme by a direct interaction with the catalytic site present in the N-domain.…”

On the Kinetic and Allosteric Regulatory Properties of the ADP-Glucose Pyrophosphorylase from Rhodococcus jostii: An Approach to Evaluate Glycogen Metabolism in Oleaginous Bacteria

“…The ADP-Glc PPase from R. jostii was characterized as a homotetrameric protein exhibiting similar S 0.5 values for substrates to those calculated for the enzyme from M. tuberculosis , but fivefold higher than those from the S. coelicolor enzyme, although with levels of activity in the same order of magnitude than the latter. Comparatively, the catalytic efficiency of the ADP-Glc PPase from R. jostii exhibits the lowest values with respect to homologous proteins from S. coelicolor and M. tuberculosis or even other PPases so far characterized ( Iglesias et al, 1991 ; Charng et al, 1992 ; Ballicora et al, 1995 , 2002 ; Ugalde et al, 1998 ; Uttaro et al, 1998 ; Gomez-Casati and Iglesias, 2002 ; Bosco et al, 2009 ; Asencion Diez et al, 2012 , 2013a , b , 2014 , 2015 ; Machtey et al, 2012 ; Ebrecht et al, 2015a , b ). Thus, the R. jostii ADP-Glc PPase might be an enzyme with low basal activity, as proposed for the ADP-Glc PPase from Streptococcus mutans with homotetrameric conformation (GlgC, with similar values of catalytic efficiency; Asencion Diez et al, 2013a ).…”