Biochemical Basis for Redox Regulation of Chloroplast-Localized Phosphofructokinase from Arabidopsis thaliana

Various proteins in plant chloroplasts are subject to thiol-based redox regulation, allowing light-responsive control of chloroplast functions. Most redox-regulated proteins are known to be reductively activated in the light in a thioredoxin (Trx)-dependent manner, but its regulatory network remains incompletely understood. Using a biochemical procedure, we here show that a specific form of phosphofructokinase (PFK) is a novel redox-regulated protein whose activity is suppressed upon reduction. PFK is a key enzyme in the glycolytic pathway. In Arabidopsis thaliana, PFK5 is targeted to chloroplasts and uniquely contains an insertion sequence harboring two Cys residues (Cys(152) and Cys(157)) in the N-terminal region. Redox shift assays using a thiol-modifying reagent indicated that PFK5 is efficiently reduced by a specific type of Trx, namely, Trx-f. PFK5 enzyme activity was lowered with the Trx-f-dependent reduction. PFK5 redox regulation was bidirectional; PFK5 was also oxidized and activated by the recently identified Trx-like2/2-Cys peroxiredoxin pathway. Mass spectrometry-based peptide mapping analysis revealed that Cys(152) and Cys(157) are critical for the intramolecular disulfide bond formation in PFK5. The involvement of Cys(152) and Cys(157) in PFK5 redox regulation was further supported by a site-directed mutagenesis study. PFK5 catalyzes the reverse reaction of fructose 1,6-bisphosphatase (FBPase), which is reduced and activated specifically by Trx-f. Our data suggest that PFK5 redox regulation, together with that of FBPase, constitutes a checkpoint for switching light/dark metabolism in chloroplasts.

Automated summary

Various proteins in plant chloroplasts are regulated by thiol-based redox processes for light-responsive control. A specific form of phosphofructokinase (PFK), PFK5, is a novel redox-regulated protein that is suppressed upon reduction, while also being activated through oxidization by specific Trx proteins. This redox regulation of PFK5, together with other proteins like FBPase, plays a crucial role in switching light/dark metabolism in chloroplasts.