Abstract
Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants.
Highlights
To tune cellular physiology, a number of proteins in the cell undergo several post-translational modifications
Our data indicate that (i) a specific isoform of Arabidopsis phosphoglycerate dehydrogenase (PGDH), PGDH1, forms the intramolecular disulfide bond, lowering PGDH enzyme activity; (ii) its disulfide bond is formed between Cys residues uniquely conserved in land plant PGDH; and (iii) some plastid-localized Trx subtypes can reduce and activate PGDH1
One of Arabidopsis plastidial Trx isoforms, Trx-f1, or Arabidopsis NADPH-Trx reductase C (NTRC), each of which was prepared in the form of monocysteinic variant [13], was used as bait in this experiment
Summary
A number of proteins in the cell undergo several post-translational modifications. Thiol-based redox regulation is one of such mechanisms; it controls enzyme activity by switching the oxidation/reduction states of Cys residues (e.g. formation/cleavage of disulfide bonds). A small ubiquitous protein thioredoxin (Trx) is largely responsible for the redox regulation. By using two Cys residues in this motif, Trx catalyzes a dithiol-disulfide exchange reaction with its target proteins, allowing modulation of their enzyme activities.
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