Structural Basis for the Limited Response to Oxidative and Thiol-Conjugating Agents by Triosephosphate Isomerase From the Photosynthetic Bacteria Synechocystis

Abstract

In plants, the ancestral cyanobacterial triosephosphate isomerase (TPI) was replaced by a duplicated version of the cytosolic TPI. This isoform acquired a transit peptide for chloroplast localization and functions in the Calvin-Benson cycle. To gain insight into the reasons for this gene replacement in plants, we characterized the TPI from the photosynthetic bacteria Synechocystis (SyTPI). SyTPI presents typical TPI enzyme kinetics profiles and assembles as a homodimer composed of two subunits that arrange in a (β-α)8 fold. We found that oxidizing agents diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS), do not inhibit the catalytic activity of SyTPI at concentrations required to inactivate plastidic and cytosolic TPIs from the plant model Arabidopsis thaliana (AtpdTPI and AtcTPI, respectively). The crystal structure of SyTPI revealed that each monomer contains three cysteines, C47, C127, and C176; however only the thiol group of C176 is solvent exposed. While AtcTPI and AtpdTPI are redox-regulated by chemical modifications of their accessible and reactive cysteines, we found that C176 of SyTPI is not sensitive to redox modification in vitro. Our data let us postulate that SyTPI was replaced by a eukaryotic TPI, because the latter contains redox-sensitive cysteines that may be subject to post-translational modifications required for modulating TPI's enzymatic activity.

Exploring triosephosphate isomerase diversity in photosynthetic organisms

“…It is hypothesised that the duplicated eukaryotic TPI replaced the prokaryotic TPI due to the greater redox sensitivity of eukaryotic TPIs enabling for the modulation of TPI activity in response to the reactive oxygen species which are produced during the light reactions of photosynthesis (Dumont et al, 2016, Castro-Torres et al, 2018. This hypothesis is consistent with phylogenomic studies which have detected a global expansion in the redox sensitive proteome coinciding with the endosymbiotic event that gave rise to the chloroplast (Woehle et al, 2017).…”

“…Accordingly, much of the existing knowledge on TPIs in photosynthetic organisms has come from thorough investigation of these two species (Dumont et al, 2016, López-Castillo et al, 2016, Castro-Torres et al, 2018.…”

“…Synechocystis has a single TPI (SpcTPI) for which both structural and kinetic data are available (Castro-Torres et al, 2018). A. thaliana has a cytoplasmic and a chloroplast TPI (cAthTPI and pAthTPI, respectively) in addition to a splice variant of pAthTPI which possesses a nine amino acid deletion (Chen and Thelen, 2010).…”

“…A. thaliana has a cytoplasmic and a chloroplast TPI (cAthTPI and pAthTPI, respectively) in addition to a splice variant of pAthTPI which possesses a nine amino acid deletion (Chen and Thelen, 2010). Both structural and kinetic data for cAthTPI and pAthTPI are available (Dumont et al, 2016, López-Castillo et al, 2016, Castro-Torres et al, 2018.…”

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

“…The inaccessibility of the thiol group of residue Cys127 and its tendency to be a target of glutathione are paradoxical. Most TPIs including, Zea mays TPI (ZmTPI), dissociate into unfolded monomers, suggesting the possibility that glutathionylation at residue Cys127 may occur at the unfolded state (Tellez et al ., ; Romero‐Romero et al ., ) or it may reflect a transient exposure of its thiol group to the surface, as suggested to occur for SyTPI (Castro‐Torres et al ., ). Residue AtcTPI‐Cys13 is located in the middle of loop 1 and forms part of the dimer interface by interacting with residues from loop 3 of the neighboring subunit (Figure a).…”