Insights into Glycogen Metabolism in Chemolithoautotrophic Bacteria from Distinctive Kinetic and Regulatory Properties of ADP-Glucose Pyrophosphorylase from Nitrosomonas europaea

Abstract

bNitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO 2 via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.

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 ).…”

“…To the best of our knowledge, this is the first time that lack of substrate specificity toward NTPs is shown in an ADP-Glc PPase from bacteria ( Ballicora et al, 2003 ), including actinobacteria ( Asencion Diez et al, 2012 , 2015 ). The closest example is the enzyme from Nitrosomonas europaea , although the latter showed less than 10% activity with other NTPs and its activity with GTP was almost no detectable ( Machtey et al, 2012 ). Additionally, we report herein, for the first time, an unusual activity regarding the high preference for GlcN-1P.…”

The ADP‐glucose pyrophosphorylase from Streptococcus mutans provides evidence for the regulation of polysaccharide biosynthesis in Firmicutes

“…8 and Table S1). A previous phylogenetic comparison between amino acid sequences of bacterial ADP-Glc PPases allowed differentiate a separate cluster including the enzyme from Grampositive bacteria with low G+C (Machtey et al, 2012).…”

Allosteric Control of Substrate Specificity of the Escherichia coli ADP-Glucose Pyrophosphorylase

“…Previous works reported that ADP-Glc PPase has a certain degree of promiscuity toward the substrates (Preiss et al, 1966 ; Lapp and Elbein, 1972 ; Hill et al, 1991 ; Machtey et al, 2012 ; Cereijo et al, 2016 ), but the control of this promiscuity has not been studied. Here, we analyzed the use of alternative substrates by the ADP-Glc PPase from Escherichia coli , and how the activators, fructose-1,6-bisphosphate (Fru-1,6-bisP) and pyruvate (Pyr), play a key role in substrate selection.…”