Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

Abstract

In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (β-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the redox environment in the chloroplast.

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

“…The role of TPIs in central metabolic pathways drives the conservation of this enzyme from bacteria to animal and plants. The nuclear genome of flowering plants encodes two fully functional TPIs, one cytosolic and one targeted into the chloroplast (Chen and Thelen, ; Dumont et al ., ; López‐Castillo et al ., ). Chloroplast TPI (pdTPI) arose by a gene duplication event from cytosolic TPI (cTPI) that replaced the cyanobacterial‐derived TPI (Reyes‐Prieto and Bhattacharya, ).…”

“…Also, cysteines can be reversibly modified by glutathionylation, nitrosylation, and disulfide bond formation (Zaffagnini et al ., , ). Different studies have shown that TPIs from photosynthetic organisms are targets for S ‐glutathionylation and nitrosylation (Ito et al ., ; Dixon et al ., ; Zaffagnini et al ., ; Dumont et al ., ; López‐Castillo et al ., ). However, the effect of glutathionylation differs among these TPIs.…”

“…With basis on the crystal structure of cTPI from A. thaliana (AtcTPI) it was hypothesized that S ‐glutationylation and chemical modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 would result in enzyme inactivation by promoting the formation an inactive monomeric species or by modifying the enzyme active site (López‐Castillo et al ., ). To understand how S ‐glutathionylation and ROS‐mediated modifications at residues AtcTPI‐Cys13 and AtcTPI‐Cys218 decrease AtcTPI enzymatic activity, we mutated those residues to amino acids that mimic the oxidation of cysteine (Asp) (Miyata et al ., ), conjugation with thiol‐conjugating adducts (Tyr or Lys) and to conserved amino acids.…”

“…In summary, our data show that chemical modifications at residues AtcTPI‐Cys218 and AtcTPI‐Cys13 renders inactive enzymes by two different mechanisms. We propose that cAtTPI glutathionylation may protect the enzyme from irreversible cysteine oxidation and decrease AtcTPI activity to re‐route carbon metabolism into the PPP and decrease oxidative stress by modifications of the oligomeric state and the active site of the enzyme (Dumont et al ., ; López‐Castillo et al ., ).…”

“…However, the effect of glutathionylation differs among these TPIs. For example, S ‐glutationylation in cTPI from A. thaliana produces a concomitant loss of enzymatic activity, and this reaction is reverted by the addition of reduced glutathione (GSH) or dithiothreitol (DTT) (Ito et al ., ), whereas S ‐glutationylation only induces a slight reduction in the activity of chloroplastic from Arabidopsis and Chlamydomonas (CrTPI) (Zaffagnini et al ., ; López‐Castillo et al ., ) and has a negligible effect on the enzymatic activity of the TPI from the photosynthetic bacteria Synechocystis (SyTPI) (Zaffagnini et al ., ; López‐Castillo et al ., ).…”