Carbohydrate Metabolism in Bacteria: Alternative Specificities in ADP-Glucose Pyrophosphorylases Open Novel Metabolic Scenarios and Biotechnological Tools

Abstract

We explored the ability of ADP-glucose pyrophosphorylase (ADP-Glc PPase) from different bacteria to use glucosamine (GlcN) metabolites as a substrate or allosteric effectors. The enzyme from the actinobacteria Kocuria rhizophila exhibited marked and distinctive sensitivity to allosteric activation by GlcN-6P when producing ADP-Glc from glucose-1-phosphate (Glc-1P) and ATP. This behavior is also seen in the enzyme from Rhodococcus spp., the only one known so far to portray this activation. GlcN-6P had a more modest effect on the enzyme from other Actinobacteria (Streptomyces coelicolor), Firmicutes (Ruminococcus albus), and Proteobacteria (Agrobacterium tumefaciens) groups. In addition, we studied the catalytic capacity of ADP-Glc PPases from the different sources using GlcN-1P as a substrate when assayed in the presence of their respective allosteric activators. In all cases, the catalytic efficiency of Glc-1P was 1-2 orders of magnitude higher than GlcN-1P, except for the unregulated heterotetrameric protein (GlgC/GgD) from Geobacillus stearothermophilus. The Glc-1P substrate preference is explained using a model of ADP-Glc PPase from A. tumefaciens based on the crystallographic structure of the enzyme from potato tuber. The substrate-binding domain localizes near the N-terminal of an α-helix, which has a partial positive charge, thus favoring the interaction with a hydroxyl rather than a charged primary amine group. Results support the scenario where the ability of ADP-Glc PPases to use GlcN-1P as an alternative occurred during evolution despite the enzyme being selected to use Glc-1P and ATP for α-glucans synthesis. As an associated consequence in such a process, certain bacteria could have improved their ability to metabolize GlcN. The work also provides insights in designing molecular tools for producing oligo and polysaccharides with amino moieties.

Keywords: allosterism; glucosamine-1-phosphate; glucosamine-6-phosphate; glucose-1-phosphate; promiscuity.

Figure 1

GlcN-6P as effector of bacterial ADP-glucose pyrophosphorylases (ADP-Glc PPases). Activities were measured according to the section “Materials and Methods,” using indicated effector concentrations.

Figure 2

Selectivity ratios for glucose-1-phosphate (Glc-1P) and GlcN-1P (ratio between catalytic efficiency for the use of Glc-1P over that with GlcN-1P).

Figure 3

Interaction of the glucose moiety of the product ADP-Glc with the ADP-Glc PPase from Agrobacterium tumefaciens and potato tuber. (A) Structure of the A. tumefaciens ADP-Glc PPase in which ADP-Glc has been modeled as described in the section “Materials and Methods.” In orange is the ribbon corresponding to the α-helix from residues F233 to H244. Green dashes indicate the distance from the OH (position 2) to the side chain oxygen of the E187 residue (3.1 Å) and to the backbone N of the residue G234 (4.0 Å), respectively. (B): Structure of the small subunit ADP-Glc PPase from potato tuber (PDB ID: 1YP4, subunit B) with ADP-Glc in the active site. In orange is the ribbon corresponding to the α-helix from residues F254 to S264. Green dashes indicate the distance to the O of side chain of the E197 residue (2.8 Å) and to the N of the backbone of the residue G255 (4.0 Å), respectively. (C) Structure of the small subunit ADP-Glc PPase from potato tuber (PDB ID: 1YP4, subunit A) with ADP in the active site. In orange is the ribbon corresponding to the α-helix from residues F254 to S264. Green dashes indicate the distance from the closest O of the β-phosphate of ADP to the backbone nitrogen of the S256 residue (3.3 Å) and to the backbone nitrogen of the residue G255 (3.9 Å), respectively.