UNDERSTANDING THE ALLOSTERIC TRIGGER FOR THE FRUCTOSE-1,6-BISPHOSPHATE REGULATION OF THE ADP-GLUCOSE PYROPHOSPHORYLASE FROM ESCHERICHIA COLI
by Carlos Maria Figueroa
ADP-glucose pyrophosphorylase is the enzyme responsible for the regulation of glycogen synthesis in bacteria. The enzyme N-terminal domain has a Rossmann-like fold with three neighbor loops facing the substrate ATP. In the Escherichia coli enzyme, one of those loops also faces the regulatory site containing Lys39, a residue involved in binding of the allosteric activator fructose-1,6-bisphosphate and its analog pyridoxal-phosphate. The other two loops contain Trp113 and Gln74, respectively, which are highly conserved among all the ADP-glucose pyrophosphorylases. Molecular modeling of the Escherichia coli enzyme showed that binding of ATP correlates with conformational changes of the latter two loops, going from an open to a closed (substrate-bound) form. Alanine mutants of Trp113 or Gln74 did not change apparent affinities for the substrates, but they became insensitive to activation by fructose-1,6-bisphosphate. By capillary electrophoresis we found that the mutant enzymes still bind fructose-1,6-bisphosphate, with similar affinity as the wild type enzyme. Since the mutations did not alter binding of the activator, they must have disrupted the communication between the regulatory and the substrate sites. This agrees with a regulatory mechanism where the interaction with the allosteric activator triggers conformational changes at the level of loops containing residues Trp113 and Gln74.
Keywords: Allostery mechanism; Activation signal propagation; Regulation dynamics; ADP-glucose pyrophosphorylase; Glycogen/Starch metabolism.
Abbreviations: ADP-Glc, ADP-glucose; ADP-Glc PPase, ADP-Glc pyrophosphorylase; CZE, capillary zone electrophoresis; Fru-1,6-P2, fructose-1,6-bisphosphate; Glc1P, glucose-1-phosphate; PLP, pyridoxal-phosphate.
ADP-glucose pyrophosphorylase (EC 188.8.131.52; ADP-Glc PPase) plays a key role in bacteria and plants catalyzing the rate limiting step of the biosynthesis of reserve polysaccharides, glycogen and starch, respectively (for reviews see [1-5]). A critical feature of this enzyme is that the activity is allosterically modulated by key intermediates of the major carbon and energy metabolism in every studied organism . These effector metabolites are indicators of high or low contents of carbon and energy within the cell, which explains why synthesis of storage polysaccharides in bacteria and plants is enhanced when cellular carbon and energy is in excess [2, 3]. For this reason, to properly understand the control of carbon and energy metabolism in these diverse organisms it is critical to unravel the molecular mechanism of the ADP-Glc PPase allosteric regulation. Despite the relatively abundant structural and kinetic information on the ADP-Glc PPase family, the molecular mechanism of the allosteric regulation has been completely unknown thus far.
ADP-Glc PPase catalyzes the formation of ADP-Glc and PPi from glucose-1P (Glc1P) and ATP. The reaction requires a divalent cation (Mg2+) and, although it is freely reversible in vitro, it mainly proceeds in the ADP-Glc synthesis direction within the cell [2, 3]. Based on specificity for allosteric regulators, ADP-Glc PPases have been classified in nine different groups [2, 3]. For example, in class I, fructose-1,6-bisphosphate (Fru-1,6-P2) activates the enzyme from enteric bacteria (e.g. Escherichia coli), and AMP is an inhibitor [2, 3], whereas 3‑phosphoglycerate activates the enzyme from plants (class VIII) and orthophosphate is an inhibitor . In all cases, these enzymes are tetramers, but there are differences between bacteria and plants. ADP-Glc PPase from E. coli is a homotetramer (α4), with subunits of ~50 kDa , whereas the enzyme from plants are heterotetramers (α2β2) of similar molecular mass .
Two ADP-Glc PPase crystallographic structures have been recently solved: a homotetrameric (α4) form from potato tuber  and the Agrobacterium tumefaciens  enzyme. In both cases, the three-dimensional structure corresponds to a sulfate-bound, allosterically inhibited form of the enzyme, which has limited the complete understanding of the enzyme’s regulatory mechanism [6, 7]. Two domains are evident: the N-terminal domain is catalytic and resembles a dinucleotide-binding Rossmann fold, whereas the C-terminal domain is involved in cooperative allosteric regulation and oligomerization [2, 6, 8-10]. Studies performed by using different experimental approaches have shown the existence of an interaction between both domains [11-14]. Current information suggests that the communication between those two domains is important but no detail has been described at the atomic level.
In this work, we developed a molecular model of the E. coli ADP-Glc PPase that strongly suggests a mechanism for propagation of the allosteric activation, in which the hydrogen bond interactions between the loops containing Gln74 and Trp113 play a critical role. After site-directed mutagenesis of those conserved residues, we obtained enzyme forms defective to activation by Fru-1,6-P2 despite the fact that the amino acids lie in a region distant from the activator binding domain. Characterization of the mutant enzymes highlights the interaction between catalytic and regulatory regions, and provides important evidence of conformational changes related with the mechanism of allosteric activation.
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