The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.

The ADP-glucose pyrophosphorylase from Streptococcus mutans provides evidence for the regulation of polysaccharide biosynthesis in Firmicutes.

Streptococcus mutans is the leading cause of dental caries worldwide. The bacterium accumulates a glycogen-like internal polysaccharide, which mainly contributes to its carionegic capacity. S.mutans has two genes (glgC and glgD) respectively encoding putative ADP-glucose pyrophosphorylases (ADP-Glc PPase), a key enzyme for glycogen synthesis in most bacteria. Herein, we report the molecular cloning and recombinant expression of both genes (separately or together) followed by the characterization of the respective enzymes. When expressed individually GlgC had ADP-Glc PPase activity, whereas GlgD was inactive. Interestingly, the coexpressed GlgC/GlgD protein was one order of magnitude more active than GlgC alone. Kinetic characterization of GlgC and GlgC/GlgD pointed out remarkable differences between them. Fructose-1,6-bis-phosphate activated GlgC by twofold, but had no effect on GlgC/GlgD. Conversely, phospho-enol-pyruvate and inorganic salts inhibited GlgC/GlgD without affecting GlgC. However, in the presence of fructose-1,6-bis-phosphate GlgC acquired a GlgC/GlgD-like behaviour, becoming sensitive to the stated inhibitors. Results indicate that S. mutans ADP-Glc PPase is an allosteric regulatory enzyme exhibiting sensitivity to modulation by key intermediates of carbohydrates metabolism in the cell. The particular regulatory properties of the S.mutans enzyme agree with phylogenetic analysis, where GlgC and GlgD proteins found in other Firmicutes arrange in distinctive clusters.