Chloroplast fructose‐1,6‐bisphosphatase: Modification of non‐covalent interactions promote the activation by chimeric Escherichia coli thioredoxins

Abstract

Although all thioredoxins contain a highly conserved amino acid sequence responsible for thiolldisulfide exchanges, only chloroplast thioredoxin-f is effective in the reductive stimulation of chloroplast fructose-l,6-bisphosphatase. We set out to determine whether Escherichia coli thioredoxin becomes functional when selected modulators alter the conformation of the target enzyme. Wild type and chimeric Escherichia coli thioredoxins match the chloroplast counterpart when the activation of chloroplast fructose-l,6-bisphosphatase is performed in the presence of fructose 1,6-bisphosphate, Ca 2+, and either trichloroacetate or 2-propanol. These modulators of enzyme activity do change the conformation of chloroplast fructose-l,6-bisphosphatase whereas bacterial thioredoxins remain unaltered. Given that fructose 1,6-bisphosphate, Ca 2+, and non-physiological perturbants modify non-covalent interactions of the protein but do not participate in redox reactions, these results strongly suggest that the conformation of the target enzyme regulates the rate of thioi/disulfide exchanges catalyzed by protein disulfide oxidoreductases.

Activation of the Potato Tuber ADP-glucose Pyrophosphorylase by Thioredoxin

“…Thioredoxin f proved to be to be more efficient than thioredoxin m in the activation process, but the difference was only 2-fold. Although other enzymes regulated by thioredoxin seem to be far more selective toward one or another of the two forms, f or m (20), in some cases it has been shown that the inclusion of enzyme effectors diminished the selectivity significantly (21). The A 0.…”

“…In the amyloplast, the main carbon source, translocated from the cytosol, is either Glc-6-P or Glc-1-P (30). The NADPH produced in this light-independent process further reduces a ferredoxin form that is present in heterotrophic plastids and serve as reducing power for ferredoxin-dependent enzymes (21). It has been described that the cytosolic thioredoxin h is reduced by NADPH via a NADP-thioredoxin reductase as in bacteria (NADP-thioredoxin system).…”

Role of Electrostatic Interactions on the Affinity of Thioredoxin for Target Proteins

“…Moreover, these studies led to the view that chloroplast Trx-m preferentially activates and deactivates NADP-malate dehydrogenase and glucose-6-phosphate dehydrogenase, respectively, whereas chloroplast Trx-f is highly efficient in the stimulation of key enzymes of photosynthetic CO 2 assimilation (CFBPase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde-3-P dehydrogenase). However, evidence suggests the existence of other, perhaps complementary, mechanisms that may regulate the specificity of Trx for target proteins (13)(14)(15)(16). For example, in vitro studies have shown that spinach chloroplast Trx-m and Escherichia coli Trx are indistinguishable from chloroplast Trx-f when the activation of CFBPase is performed in the presence of fructose 1,6-bisphosphate and Ca 2ϩ (13,15).…”

“…However, evidence suggests the existence of other, perhaps complementary, mechanisms that may regulate the specificity of Trx for target proteins (13)(14)(15)(16). For example, in vitro studies have shown that spinach chloroplast Trx-m and Escherichia coli Trx are indistinguishable from chloroplast Trx-f when the activation of CFBPase is performed in the presence of fructose 1,6-bisphosphate and Ca 2ϩ (13,15). Native (oxidized) CFBPase serves as excellent substrate for the analysis of the interaction between Trx and target proteins because the enzyme activity is effectively modulated not only by the formation of sulfhydryl groups but also by subtle alter-ations of the tertiary structure (14,15).…”

“…For example, in vitro studies have shown that spinach chloroplast Trx-m and Escherichia coli Trx are indistinguishable from chloroplast Trx-f when the activation of CFBPase is performed in the presence of fructose 1,6-bisphosphate and Ca 2ϩ (13,15). Native (oxidized) CFBPase serves as excellent substrate for the analysis of the interaction between Trx and target proteins because the enzyme activity is effectively modulated not only by the formation of sulfhydryl groups but also by subtle alter-ations of the tertiary structure (14,15). The reductive activation of CFBPase, but not the catalytic step, depends on three cysteines clustered in a solvent-exposed structure encompassing ϳ15 amino acid residues (named the 170's loop by Villeret et al) (17)(18)(19).…”