Leonardo Astolfi Rosado

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Self-protection of cytosolic malate dehydrogenase against oxidative stress in Arabidopsis

Plant malate dehydrogenase (MDH) isoforms are found in different cell compartments and function in key metabolic pathways. It is well known that the chloroplastic NADP-dependent MDH activities are strictly redox regulated and controlled by light. However, redox dependence of other NAD-dependent MDH isoforms have been less studied. Here, we show by in vitro biochemical characterization that the major cytosolic MDH isoform (cytMDH1) is sensitive to H2O2 through sulfur oxidation of cysteines and methionines. CytMDH1 oxidation affects the kinetics, secondary structure, and thermodynamic stability of cytMDH1. Moreover, MS analyses and comparison of crystal structures between the reduced and H2O2-treated cytMDH1 further show that thioredoxin-reversible homodimerization of cytMDH1 through Cys330 disulfide formation protects the protein from overoxidation. Consistently, we found that cytosolic thioredoxins interact specifically with cytMDH in a yeast two-hybrid system. Importantly, we also show that cytosolic and chloroplastic, but not mitochondrial NAD-MDH activities are sensitive to H2O2 stress in Arabidopsis. NAD-MDH activities decreased both in a catalase2 mutant and in an NADP-thioredoxin reductase mutant, emphasizing the importance of the thioredoxin-reducing system to protect MDH from oxidation in vivo. We propose that the redox switch of the MDH activity contributes to adapt the cell metabolism to environmental constraints.

Disulfide bond formation protects Arabidopsis thaliana glutathione transferase tau 23 from oxidative damage.

BACKGROUND Glutathione transferases play an important role as detoxifying enzymes. In A. thaliana, elevated levels of reactive oxygen species (ROS), provoked during biotic and abiotic stress, influence the activity of GSTU23. The aim of this study is to determine the impact of oxidative stress on the function and structure of GSTU23. METHODS The impact of oxidation on the function of GSTU23 was studied using a glutathione transferase biochemical assay and mass spectrometry. With kinetics, circular dichroism and thermodynamics, we compared reduced with oxidized GSTU23. X-ray crystal structures of GSTU23 visualize the impact of oxidation on methionines and cysteines. RESULTS In the presence of 100μM H2O2, oxidation of the methionine side-chain to a sulfoxide is the prominent post-translational modification, which can be reduced by C. diphtheriae MsrA and MsrB. However, increasing the level to 200μM H2O2 results in a reversible intramolecular disulfide between Cys65-Cys110, which is substrate for glutaredoxin. Under these oxidizing conditions, GSTU23 undergoes a structural change and forms a more favourable enzyme-substrate complex to overcome kcat decrease. CONCLUSIONS AND SIGNIFICANCE At lower H2O2 levels (100μM), GSTU23 forms methionine sulfoxides. Specifically, oxidation of Met14, located near the catalytic Ser13, could interfere with both GSH binding and catalytic activation. At higher H2O2 levels (200μM), the Cys65-Cys110 disulfide bond protects other cysteines and also methionines from overoxidation. This study shows the impact of oxidative stress on GSTU23 regulated by methionine sulfoxide reductases and glutaredoxin, and the mechanisms involved in maintaining its catalytic functionality under oxidizing conditions.

The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S -mycothiolation under oxidative stress

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.

Redox-regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9 induces H-site flexibility: How Plant Phi 9 GST Survives Oxidative Stress

Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X‐ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH‐binding site (G‐site) and a hydrophobic co‐substrate‐binding site (H‐site). At elevated H2O2 concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H‐site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox‐regulated enzyme.

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis

The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol–disulfide mechanism. We also found that Rv2466c uses a monothiol–disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA–mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.

Protein promiscuity in H 2 O 2 signaling

Abstract Significance: Decrypting the cellular response to oxidative stress relies on a comprehensive understanding of the redox signaling pathways stimulated under oxidizing conditions. Redox sign...

Protein Promiscuity in H2O2 Signaling

Abstract Significance: Decrypting the cellular response to oxidative stress relies on a comprehensive understanding of the redox signaling pathways stimulated under oxidizing conditions. Redox sign...