Crystal structures of glycogen-debranching enzyme mutants in complex with oligosaccharides

Debranching is a critical step in the mobilization of the important energy store glycogen. In eukaryotes, including fungi and animals, the highly conserved glycogen-debranching enzyme (GDE) debranches glycogen by a glucanotransferase (GT) reaction followed by a glucosidase (GC) reaction. Previous work indicated that these reactions are catalyzed by two active sites located more than 50 Å apart and provided insights into their catalytic mechanisms and substrate recognition. Here, five crystal structures of GDE in complex with oligosaccharides with 4–9 glucose residues are presented. The data suggest that the glycogen main chain plays a critical role in binding to the GT and GC active sites of GDE and that a minimum of five main-chain residues are required for optimal binding.

Streptococcus infantarius 25124 isolated from pozol produces a high molecular weight amylopullulanase, a key enzyme for niche colonization

Pozol is a beverage made with maize dough that is prepared after boiling the kernels in limewater, causing a decrease in soluble sugars, with starch being the main fermentable carbohydrate in the dough. Previously, Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) was identified as the most amylolytic bacteria isolated in this product. Analysis of Sii-25124 amylolytic enzymes revealed two amylases, a cytoplasmic α-amylase of 55.7 kDa and an extracellular amylopullulanase of 246.3 kDa, with two catalytic domains, one typical of an α-amylase and another typical of a pullulanase/glycogen debranching enzyme. Characterization of the joint activity of both enzymes using Sii-25124 cell lysate supernatant demonstrated stability between 30 °C and 45°C, and pH stability in a range between 6.8 and 8.0. The joint activity of Sii-25124 amylases showed a fast production of reducing sugars when starch was used as the substrate. In contrast, reducing sugar production from amylopectin was lower, but it steadily increased throughout the reaction time. The amylopullulanase produced by Sii-25124 hydrolyzes the starch in the dough to produce low molecular weight oligosaccharides, which may be transported into Sii-25124 cells, so that intracellular α-amylase hydrolyzes them to mono- and disaccharides. Amylopullulanase production by Sii-25124 could be an example of a specialized enzyme that successfully dominates starchy food fermentation.

Disruption of glycogen metabolism alters cell size in Escherichia coli

The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation - glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) - were disrupted, we examined if endogenous polyglucan degradation affects cell size. It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ΔmalP mutant cells did, however, accumulate increased DnaA amounts at late growth stages indicating a potential effect on DNA replication. Replication run-out experiments demonstrated that this was indeed the case, and that DNA replication was also affected in the other mutants. Bacteria with a disruption in glgX accumulated glycogen and protein inclusion bodies that coincided with each other at inter-nucleoid and polar regions.

Expression of a Functional Recombinant Human Glycogen Debranching Enzyme (hGDE) in N. benthamiana Plants and in Hairy Root Cultures

Background: Glycogen storage disease type III (GSDIII, Cori/Forbes disease) is a metabolic disorder due to the deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (about 176 kDa) with two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Several mutations along the amylo-alpha-1,6-glucosidase,4-alphaglucanotransferase (Agl) gene are associated with loss of enzymatic activity. The unique treatment for GSDIII, at the moment, is based on diet. The potential of plants to manufacture exogenous engineered compounds for pharmaceutical purposes, from small to complex protein molecules such as vaccines, antibodies and other therapeutic/prophylactic entities, was shown by modern biotechnology through “Plant Molecular Farming”. Objective/method: In an attempt to develop novel protein-based therapeutics for GSDIII, the Agl gene, encoding for the human GDE (hGDE) was engineered for expression as a histidinetagged GDE protein both in Nicotiana benthamiana plants by a transient expression approach, and in axenic hairy root in vitro cultures (HR) from Lycopersicum esculentum and Beta vulgaris. Results: In both plant-based expression formats, the hGDE protein accumulated in the soluble fraction of extracts. The plant-derived protein was purified by affinity chromatography in native conditions showing glycogen debranching activity. Conclusion: These investigations will be useful for the design of a new generation of biopharmaceuticals based on recombinant GDE protein that might represent, in the future, a possible therapeutic option for GSDIII.