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.
Highlights
Triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP)
Since the crystal structures of TPIs are similar between species we focused on the position of cysteine residues that can function as regulatory elements
The role of cysteines located in structural positions equivalent to cTPI-C13 and C127 has been widely studied in various organisms, mainly in relationship to enzymatic activity and protein stability (Reyes-Vivas et al, 2001; Maithal et al, 2002; Gonzalez-Mondragon et al, 2004; Samanta et al, 2011)
Summary
Triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). The structure of this enzyme consists of eight alternate βstrands and α-helices, dubbed TIM or (β/α) barrel fold (Banner et al, 1975). Crystal structures of more than 40 TPIs from different species are deposited in the Protein Data Bank (PDB). One or various cytoplasmic and chloroplast TPIs (cTPI and pdTPI), are present in plant genomes. In unicellular green algae, the first reactions of the glycolytic pathway from glucose phosphorylation to triosephosphate isomerization occur inside the chloroplast (reviewed in Johnson and Alric, 2013) and unicellular green algae only contain one TPI isoform
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