Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson–Calvin cycle enzyme enslaved to its cysteine residues

Abstract

Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson-Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK-GAPDH-CP12 complex. These results uncover a new mechanism for the PRK-GAPDH-CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

Storage Compound Accumulation in Diatoms as Response to Elevated CO2 Concentration

“…In P. tricornutum, the formation of the regulatory complex GAPDH/PRK/CP12 has been already discarded [66], and there is no evidence of its formation in T. pseudonana. Moreover, it has been shown that the formation of this ternary complex requires the presence of two cysteine residues on PRK, that are absent in diatom PRK [67]. This suggests that regulation of GAPDH in these two diatoms relies on a different mechanism.…”

Interaction between Silver Nanoparticles and Two Dehydrogenases: Role of Thiol Groups

“…Circular Dichroism : The GAPDH and MDH were diluted to 5 mg L −1 (35 and 75 × 10 −6 m for tetrameric GAPDH and dimeric MDH, respectively) and placed into a quartz cuvette with a final concentration at 8 or 80 mg L −1 AgNPs, or 8 mg L −1 Ag + , or 46 mg L −1 PVP. CD spectra from 190 to 250 nm were recorded on a spectropolarimeter (Jasco J‐1100, Jasco Analytical Instruments, Easton, Maryland) at 25 °C . For each spectrum, there were six successive scans.…”

“…CD spectra from 190 to 250 nm were recorded on a spectropolarimeter (Jasco J-1100, Jasco Analytical Instruments, Easton, Maryland) at 25 °C. [34] For each spectrum, there were six successive scans. The mean residue molar ellipticity θ (deg cm 2 dmol −1 ) was calculated from raw ellipticity E (mdeg) using Equation (1)…”

Crystal structure of phosphoribulokinase fromSynechococcussp. strain PCC 6301

“…The structural basis for this reversible inactivation is unknown. Sequences from -cyanobacteria, green algae and plants contain two pairs of conserved cysteine residues: one close to the presumed ATP-binding pocket and one close to the C-terminus (Thieulin-Pardo et al, 2015). Whereas thiol modification of Cys16 (but not the C16S mutation) of the N-terminal pair in PRK from green algae and plants strongly impairs the enzyme activity, modification or mutation of the C-terminal pair has no effect on enzyme activity in vitro (Milanez et al, 1991;Thieulin-Pardo et al, 2015).…”

“…Sequences from -cyanobacteria, green algae and plants contain two pairs of conserved cysteine residues: one close to the presumed ATP-binding pocket and one close to the C-terminus (Thieulin-Pardo et al, 2015). Whereas thiol modification of Cys16 (but not the C16S mutation) of the N-terminal pair in PRK from green algae and plants strongly impairs the enzyme activity, modification or mutation of the C-terminal pair has no effect on enzyme activity in vitro (Milanez et al, 1991;Thieulin-Pardo et al, 2015). Oxidized PRK from these organisms has been reported to become inactive (Porter et al, 1990) and to subsequently bind another CBB cycle enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in a process which is mediated by the small intrinsically disordered protein CP12 in a redoxdependent manner (Clasper et al, 1991;Howard et al, 2008;Thieulin-Pardo et al, 2015;Avilan et al, 1997;Lebreton et al, 1997).…”