AUTHOR=Gurrieri Libero , Distefano Luca , Pirone Claudia , Horrer Daniel , Seung David , Zaffagnini Mirko , Rouhier Nicolas , Trost Paolo , Santelia Diana , Sparla Francesca 

TITLE=The Thioredoxin-Regulated α-Amylase 3 of Arabidopsis thaliana Is a Target of S-Glutathionylation

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.00993

DOI=10.3389/fpls.2019.00993

ISSN=1664-462X

ABSTRACT=<p>Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, <italic>At</italic>AMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that <italic>At</italic>AMY3 activity was sensitive to ROS, such as H<sub>2</sub>O<sub>2</sub>. Treatments with H<sub>2</sub>O<sub>2</sub> inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized <italic>At</italic>AMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated <italic>At</italic>AMY3 and performing oxidation experiments of Cys-to-Ser variants of <italic>At</italic>AMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the p<italic>K</italic><sub>a</sub> values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (p<italic>K</italic><sub>a1</sub> = 5.70 ± 0.28; p<italic>K</italic><sub>a2</sub> = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of <italic>At</italic>AMY3 is proposed.</p>