Conserved residues of the Pro103-Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase.

Bacteria and plants produce glycogen and starch, respectively, as their primary storage polysaccharides. The synthesis of these polymers occurs with the donation of a glucose moiety from ADP-glucose (ADP-Glc) to an existing α-1,4-polyglucan chain. It is the production of ADP-Glc, catalyzed by the enzyme ADP-glucose pyrophosphorylase (ATP:α-D-glucose-1-phosphate adenylyltransferase; EC 2.7.7.27; ADP-Glc PPase), that acts as the rate limiting step in glycogen and starch production.1 This Mg2+ dependent reaction proceeds with ATP binding first to the enzyme, followed by glucose-1-phosphate (Glc1P), with the subsequent production of ADP-Glc and pyrophosphate.2,3 All known plant and bacterial ADP-Glc PPases are derived from a common ancestor and are predicted to share considerable structural similarities.4,5 These structural features are evident in the crystallographic structures of the potato tuber6 and Agrobacterium tumefaciens7 enzymes. Notably, the enzymes are tetramers with ∼50 kDa subunits. Enteric and cyanobacterial enzymes are homotetrameric (α4), whereas those of Firmicutes, unicellular algae, and higher plants are heterotetrameric (α2β2).4,5 In the latter, the two subunit types have similar structure and share a common ancestry, although it is sometimes the case that just one subunit type is catalytic.8,9 In most organisms, this enzyme is allosterically regulated by intermediates of the main carbon assimilatory pathway that exist within that species.4,5,10 In the case of E. coli, glycolysis is the major pathway and fructose-1,6-bisphosphate (FBP) is the primary activator of the E. coli ADP-Glc PPase.4 Of the ADP-Glc PPases that have been characterized, the E. coli enzyme has some of the largest changes in basal activity brought about by the activator. For instance, it increases specific activity and ATP affinity by at least 25- and 15-fold, respectively.11,12 Previous mutagenesis studies involving ADP-Glc PPases have provided some information regarding the allosteric activation mechanism of these enzymes. For example, studies on chimeric ADP-Glc PPases have shown that the C-terminal domain is important for allosteric effector specificity.12 Some alanine mutants of N-terminal arginine residues in the A. tumefaciens enzyme had altered allosteric responses, such as R32A which had significantly reduced affinity for the activator fructose-6-P.13 Several N-terminal residues in the E. coli ADP-Glc PPase have also been associated with allosteric activation. Lys39 is part of the FBP binding site,14 and Gln74, which is universally conserved amongst ADP-Glc PPases, is needed in order for FBP to exhibit regulatory role.11 The alanine mutant of the nearly universally conserved Trp113 had a similar kinetic profile to Q74A, even though FBP still binds to these enzymes.11 W116A and Q75A mutants of the potato tuber enzyme also fail to respond to allosteric activator.15 A model of the E. coli ADP-Glc PPase that highlights these regions is shown in Figure 1. Despite all of these data, it is not known how the allosteric signal is transmitted and what specific interactions are involved. Figure 1 Model of the E. coli ADP-Glc PPase subunit, shown in cartoon representation. For reference, the Pro103–Arg115 loop is colored red, the C-terminal domain is in orange, Asn38–Ala40 are colored blue, Thr73–Gln75 are yellow, Leu25–Gly30 ... Here, we use molecular dynamics (MD) to get insight into what residues are involved in an allosteric pathway in the E. coli ADP-Glc PPase. Allosteric effectors may alter communication pathways that exist within an enzyme.16 For that reason, correlated movement analysis, which examines cooperative motions within a protein, may be a useful tool for elucidating such interactions.17 However, because this technique does not consider the physical proximity of correlated regions, further analysis may be needed to identify spatially connected communication pathways. To that end, a network pathway analysis, which accounts for the physical connectivity of correlated regions, can help pinpoint such tracts.18 These techniques, along with an examination of some hydrogen bond networks and structural comparisons with other ADP-Glc PPases, suggested that the span of residues from Pro103 to Arg115 is important in an allosteric activation pathway in the E. coli enzyme. We tested this hypothesis by performing alanine scanning mutagenesis on the residues of this loop and kinetically characterizing the mutants, several of which were distinguished by a disruption of the allosteric response. In addition to this, sequence alignments of known ADP-Glc PPases that focused on this loop region, as well as structural alignments of different bacterial NDP-glucose pyrophosphorylases (NDP-Glc PPases), provided insight into the evolution of allosteric regulation in ADP-Glc PPases.