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.

Comparative analysis between two GT4 glycosyltransferases related to polysaccharide biosynthesis inRhodococcus jostiiRHA1

“…Still, kinetic values suggest that GlcN-1P might be metabolically relevant. In this context, the high efficiency of RjoGlgAc towards GlcN-1P reinforces the putative importance of this metabolite, being directed to yet unknown intracellular fates, different for being just a mere intermediary between central metabolism and peptidoglycan synthesis/degradation [20,29,30]. Worthy to note the RjoGlgAc apparent affinity for GlcN-1P is in the same millimolar order as that of the rhodococcal GlgCs for the same substrate, as reported earlier [29].…”

“…Worthy to note the RjoGlgAc apparent affinity for GlcN-1P is in the same millimolar order as that of the rhodococcal GlgCs for the same substrate, as reported earlier [29]. In addition, we extended the study to other bacterial ADP-GlcPPases and determined that GlcN-1P acts as a secondary yet efficient substrate in most cases, concomitantly with activation by GlcN-6P [30]. Taken together, we are tempted to speculate that in certain organisms (such as R. jostii) and conditions, GlcN might find a way to be incorporated into different molecules.…”

“…pyrophosphorylase RjoGalU2 (~59 mM -1 seg -1 ) [20] and is higher than those obtained for rhodococcal ADP-GlcPPases, which are around 0.6 mM -1 seg -1 , after activation by Glc-6P and/or GlcN-6P [29,30]. These efficiency parameters suggest a metabolic plausibility [64].…”

High Fructose Causes More Prominent Liver Steatohepatitis with Leaky Gut Similar to High Glucose Administration in Mice and Attenuation by Lactiplantibacillus plantarum dfa1

“…The high abundance of carbohydrates in the diet produces gut dysbiosis [ 28 ] (an alteration of organisms in the intestinal tract [ 29 ]), in part due to the different abilities in carbohydrate metabolism between different groups of bacteria [ 30 , 31 ]. The increase in gut mucosal damage means that high MW molecules, such as LPS, can be directly translocated into the liver and circulatory system [ 32 , 33 ] leading to more severe steatohepatitis and systemic inflammation, respectively.…”

High Fructose Causes More Prominent Liver Steatohepatitis With Leaky Gut Similar to High Glucose Administration in Mice and Attenuation by Lactiplantibacillus plantarum dfa1

“…The high abundance of carbohydrates in the diet produces gut dysbiosis [28] (an alteration of organisms in the intestinal tract [29]), in part due to different abilities in carbohydrate metabolism between different groups of bacteria [30,31]. The increase in gut mucosal damage in diabetes and obesity that high-MW molecules, such as LPS (referred to as metabolic endotoxemia), can be directly translocated into the liver and circulatory system [32,33], leading to more severe steatohepatitis and systemic inflammation, respec-3 of 25 tively.…”