Thioredoxin (Trx)-mediated redox regulation is a posttranslational mechanism that controls enzyme activity by reversibly switching the oxidation/reduction states of Cys residues. In plant cells, numerous enzymes across diverse biological systems have been suggested as targets of redox regulation; however, a complete understanding is lacking. In this study, we report that phosphoglucose isomerase (PGI) in plastids represents a novel class of redox-sensitive enzymes. PGI catalyzes the reversible interconversion of fructose 6-phosphate and glucose 6-phosphate and operates at the branch point between the Calvin-Benson cycle and the starch synthesis pathway in plastids. Using an affinity chromatography-based method, we found that plastidial PGI physically interacts with Trx in a redox-dependent manner. In vitro assays with recombinant proteins from Arabidopsis thaliana revealed that plastidial, but not cytosolic, PGI forms an intramolecular disulfide bond. Among plastid-localized Trx subtypes, the f- and m-types were more effective in reductively cleaving the disulfide bond. MS-based peptide mapping, site-directed mutagenesis, and structural modeling identified the redox-active Cys pair. Furthermore, in vivo analysis using Arabidopsis leaves showed that plastidial PGI is converted from oxidized to reduced states upon illumination, which absolutely depends on the Trx system. Notably, despite these redox modifications, PGI catalytic activity remained nearly identical in both states. Although PGI activity was affected by some metabolites and pH, it showed no sensitivity to redox state. Our findings demonstrate that plastidial PGI is a redox-sensitive enzyme but functionally uncoupled from activity modulation.
Calmodulin (CaM) serves an essential role in eukaryotic cells as a Ca2+ sensor. Ca2+ binding leads to conformation changes in CaM that enable engagement of a repertoire of enzymes and the regulation of their catalytic activities. Classically, Ca2+-CaM binds to an inhibitory pseudosubstrate sequence C-terminal to the kinase domain in members of the Ca2+-CaM-dependent protein kinase (CAMK) family and relieves inhibition to promote catalytic activity. Here, we report an unexpected mechanism by which CaM can bind CHK2 kinase to inhibit its kinase activity. Using biochemical, biophysical and structural mass spectrometry, we identify a direct interaction of Ca2+-CaM with the CHK2 kinase domain that suppresses CHK2 catalytic activity in vitro and identify K373 in CHK2 as crucial for cell proliferation in human cells following DNA damage. Our findings add direct suppression of kinase activity to the repertoire of CaM's functions, complementing the paradigmatic mechanism of promoting kinase activity through autoinhibitory domain sequestration.
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