The Firmicutes Case on the Regulation of Bacterial ADP-Glucose Pyrophosphorylase

CEREIJO AE; IGLESIAS AA; DEMONTE AM; ASENCION DIEZ MD; BALLICORA MA

Chicago

Congreso; American Society for Biochemistry and Molecular Biology (ASBMB) General Meeting 2017; 2017

American Society for Biochemistry and Molecular Biology (ASBMB)

ADP-glucose pyrophosphorylase (EC 2.7.7.27) catalyzes the key step in the prokaryotic pathway for glycogen synthesis. The enzyme from Firmicutes is encoded by two genes, glgC and glgD, leading to a heterotetrameric protein structure unlike other bacterial ADP-glucose pyrophosphorylases. The enzyme from different orders (Bacillales, Clostridiales and Lactobacillales) in the Firmicutes phylum presents dissimilar kinetic and regulatory properties, as shown in this work. Thus, we comparatively studied the ADP-glucose pyrophosphorylases from Ruminococcus albus (Clostridiales), Streptococcus mutans (Lactobacillales) and Geobacillus stearothemophilus (Bacillales) to shed light on the structure-to-function relationships of the firmicute enzyme. In each case, we produced separate GlgC or GlgD subunits, as well as the GlgC/GlgD co-expressed forms. There were two active quaternary structures, homotetrameric GlgC and heterotetrameric GlgC/GlgD, whereas GlgD by itself was inactive for all species tested. Nevertheless, the presence of the GlgD in the co-expressed enzymes depicted different kinetic and regulatory effects depending on the source, which suggests distinct evolutionary adaptations for the non-catalytic subunit in each of the Firmicutes orders. For example, in the S. mutans, GlgC/GlgD was one order of magnitude more active than GlgC alone. In the others, the activity of GlgC/GlgD was 2-fold higher than the respective GlgC. Two effectors (fructose-1,6-bisphosphate and phosphoenolpyruvate), opposing to the insensitive enzyme from G. stearotherophilus, modulated the S. mutans and R. albus ADP-glucose pyrophosphorylases. Interestingly, phosphoenolpyruvate is a highly specific inhibitor of GlgC/GlgD ADP-glucose pyrophosphorylase from S. mutans but an activator of both GlgC and GlgC/GlgD R. albus enzymes. The latter ones were also inhibited by fructose-1,6-bisphosphate, whereas this metabolite activated the S. mutans GlgC conformation. Thus, results suggest that allosteric control might be a common feature rather than an exception in the ADP-glucose pyrophosphorylase from Firmicutes. Consequently, Glycogen synthesis may be regulated by metabolites from the main carbon utilization pathway, as reported in other bacteria.