Structural Basis of Glycogen Biosynthesis Regulation in Bacteria

Abstract

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.

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

“…ADP-Glc PPases have a common catalytic domain with other sugar nucleotide pyrophosphorylases (Jin et al, 2005; Cupp-Vickery et al, 2008; Cifuente et al, 2016). The formers generally have an extended C-terminal domain (120 to 150 amino acids) and a slightly longer N-terminal domain (10–40 amino acids).…”

The allosteric control mechanism of bacterial glycogen biosynthesis disclosed by cryoEM

“…[11][12][13][14] AGPase catalyzes the reversible condensation reaction between adenosine triphosphate (ATP) and glucose 1-phosphate (G1P) to produce ADP-glucose and pyrophosphate (PPi; Figure 1C). 5,15,16,17 Specifically, the oxygen on the phosphate group of G1P acts as a nucleophile attacking the α-PO 4 group of the nucleoside triphosphate, leading to the liberation of PPi ( Figure S1). 18 The reaction is held in the presence of the divalent metal cation Mg 2+ , which minimizes the charge repulsion between phosphate groups, favoring nucleophile activation.…”

“…26,27,28,29 The two subunits have different functions; α is the catalytic subunit, whereas β is the regulatory subunit. To date, three crystal structures of AGPases have been reported, those of the bacterial homotetrameric AGPases from Escherichia coli (EcAGPase) 5,16,30 and Agrobacterium tumefaciens (AtAGPase), 31,32 and a recombinant homotetrameric version of the small subunit (α4) of the photosynthetic potato tuber AGPase (StAGPase). 33 The activity of the paradigmatic EcAGPase is enhanced by the high-energy glycolytic intermediate fructose-1,6-bisphosphate (FBP), whereas, the ubiquitous low-energy metabolite AMP inhibits the enzyme.…”

“…[34][35][36][37] We reported the first crystal structures of the paradigmatic EcAGPase in complex with FBP and AMP, respectively. 16,30 These structures provided a first perspective of the regulatory sites and the identification of secondary structure elements likely participating into the allosteric regulation. However, the structural data prohibited the visualization of the architecture of EcAGPase in biologically relevant conformations and the comprehension on how the allosteric regulators act in a concerted manner to control the enzymatic activity.…”

“…However, the organization of the protein molecules inside the crystal packing prevents, in many cases, the visualization of native biologically relevant conformational states associated to the binding of substrates and regulatory molecules, impeding the construction of accurate models to explain allosteric regulation. 16,[31][32][33] In this work, we disclose the cryoEM structures of the homotetrameric EcAGPase (a ca. 200 kDa enzyme, with each protomer of 48.7 kDa), in complex with FBP and AMP, both at 3.0 Å resolution, respectively.…”