Joris Messens

Structure, function and mechanism of thioredoxin proteins

Thioredoxins are ubiquitous antioxidant enzymes that play important roles in many health-related cellular processes. As such, the fundamental knowledge of how these enzymes work is of prime importance for understanding cellular redox mechanisms and for laying the ground for the development of future therapeutic approaches. Over the past 40 years, a really impressive amount of data has been published on thioredoxins. Here, we review the most significant results that have contributed to our knowledge regarding the structure, the function, and the mechanism of these crucial enzymes.

Protein sulfenic acid formation: From cellular damage to redox regulation

Protein sulfenic acid formation has long been regarded as unwanted damage caused by reactive oxygen species (ROS). However, over the past 10 years, accumulating evidence has shown that the reversible oxidation of cysteine thiol groups to sulfenic acid functions as a redox-based signal transduction mechanism. Here, we review the mechanisms of sulfenic acid formation by ROS. We present some of the most important roles played by sulfenic acids in living cells as well as the pathways that regulate sulfenic acid formation. We highlight the experimental tools that have been developed to study the cellular sulfenome and show how computational approaches might help to better understand the mechanisms of sulfenic acid formation.

A periplasmic reducing system protects single cysteine residues from oxidation.

Periplasmic Redox Regulation The oxidation state of intracellular and extracellular proteins are carefully managed by cellular redox machineries. Depuydt et al. (p. 1109) discovered a reducing system that protects single cysteine residues from oxidation in the bacterial periplasm. DsbG, a thioredoxin-related protein, appears to be a key player in that system and is the first reductase identified in the periplasm of Escherichia coli. Together with DsbC, DsbG controls the global sulfenic acid content of this compartment. Sulfenic acid formation is a major posttranslational modification in the periplasm, and three homologous L,D-transpeptidases are substrates of DsbG. Sulfenic acid formation is not restricted to E. coli, but is ubiquitous. Because proteins from the thioredoxin superfamily are widespread, similar thioredoxin-related proteins may control cellular sulfenic acid more widely. A thioredoxin-like enzyme controls the oxidation state of the bacterial periplasm. The thiol group of the amino acid cysteine can be modified to regulate protein activity. The Escherichia coli periplasm is an oxidizing environment in which most cysteine residues are involved in disulfide bonds. However, many periplasmic proteins contain single cysteine residues, which are vulnerable to oxidation to sulfenic acids and then irreversibly modified to sulfinic and sulfonic acids. We discovered that DsbG and DsbC, two thioredoxin-related proteins, control the global sulfenic acid content of the periplasm and protect single cysteine residues from oxidation. DsbG interacts with the YbiS protein and, along with DsbC, regulates oxidation of its catalytic cysteine residue. Thus, a potentially widespread mechanism controls sulfenic acid modification in the cellular environment.

How Proteins Form Disulfide Bonds

The identification of protein disulfide isomerase, almost 50 years ago, opened the way to the study of oxidative protein folding. Oxidative protein folding refers to the composite process by which a protein recovers both its native structure and its native disulfide bonds. Pathways that form disulfide bonds have now been unraveled in the bacterial periplasm (disulfide bond protein A [DsbA], DsbB, DsbC, DsbG, and DsbD), the endoplasmic reticulum (protein disulfide isomerase and Ero1), and the mitochondrial intermembrane space (Mia40 and Erv1). This review summarizes the current knowledge on disulfide bond formation in both prokaryotes and eukaryotes and highlights the major problems that remain to be solved.

Understanding the pKa of Redox Cysteines: The Key Role of Hydrogen Bonding

Many cellular functions involve cysteine chemistry via thiol-disulfide exchange pathways. The nucleophilic cysteines of the enzymes involved are activated as thiolate. A thiolate is much more reactive than a neutral thiol. Therefore, determining and understanding the pK(a)s of functional cysteines are important aspects of biochemistry and molecular biology with direct implications for redox signaling. Here, we describe the experimental and theoretical methods to determine cysteine pK(a) values, and we examine the factors that control these pK(a)s. Drawing largely on experience gained with the thioredoxin superfamily, we examine the roles of solvation, charge-charge, helix macrodipole, and hydrogen bonding interactions as pK(a)-modulating factors. The contributions of these factors in influencing cysteine pK(a)s and the associated chemistry, including the relevance for the reaction kinetics and thermodynamics, are discussed. This analysis highlights the critical role of direct hydrogen bonding to the cysteine sulfur as a key factor modulating the equilibrium between thiol S-H and thiolate S(-). This role is easily understood intuitively and provides a framework for biochemical functional insights.

Arsenate reductase from S. aureus plasmid pI258 is a phosphatase drafted for redox duty

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 plays a role in bacterial heavy metal resistance and catalyzes the reduction of arsenate to arsenite. The structures of the oxidized and reduced forms of ArsC were solved. ArsC has the PTPase I fold typical for low molecular weight tyrosine phosphatases (LMW PTPases). Remarkably, kinetic experiments show that pI258 ArsC also catalyzes the tyrosine phosphatase reaction in addition to arsenate reduction. These results provide evidence that ArsC from pI258 evolved from LMW PTPase by the grafting of a redox function onto a pre-existing catalytic site and that its evolutionary origin is different from those of arsenate reductases from Escherichia coli plasmid R773 and from Saccharomyces cerevisiae. The mechanism proposed here for the catalysis of arsenate reduction by pI258 ArsC involves a nucleophilic attack by Cys 10 on arsenate, the formation of a covalent intermediate and the transport of oxidative equivalents by a disulfide cascade. The reaction is associated with major structural changes in the ArsC.

The fimbrial adhesin F17‐G of enterotoxigenic Escherichia coli has an immunoglobulin‐like lectin domain that binds N‐acetylglucosamine

The F17‐G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N‐acetyl‐β‐d‐glucosamine‐presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 Å resolution crystal structure of the lectin domain of F17‐G, both free and in complex with N‐acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid‐shaped protein in a conserved site around which all natural variations of F17‐G are clustered. A model is proposed for the interaction between F17‐fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17‐G binding to N‐acetylglucosamine (0.85 mM−1). Unexpectedly, the F17‐G structure reveals that the lectin domains of the F17‐G, PapGII and FimH fimbrial adhesins all share the immunoglobulin‐like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C‐terminal β‐strand G of the immunoglobulin‐like fold and provides new insights into pilus assembly, function and adhesion.

Arsenate reductase, mycothiol, and mycoredoxin concert thiol/disulfide exchange.

We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of ∼5 m-1 s-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.

The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner

In Escherichia coli, DsbA introduces disulphide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulphides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulphides in proteins with multiple cysteines. These incorrect disulphides are thought to be corrected by a protein disulphide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolated from the DsbA/DsbB pathway. We show that the DsbC and DsbA pathways are more intimately connected than previously thought. dsbA‐dsbC‐ mutants have a number of phenotypes not exhibited by either dsbA‐, dsbC‐ or dsbA‐dsbD‐ mutations: they exhibit an increased permeability of the outer membrane, are resistant to the lambdoid phage Φ80, and are unable to assemble the maltoporin LamB. Using differential two‐dimensional liquid chromatographic tandem mass spectrometry/mass spectrometry analysis, we estimated the abundance of about 130 secreted proteins in various dsb‐ strains. dsbA‐dsbC‐ mutants exhibit unique changes at the protein level that are not exhibited by dsbA‐dsbD‐ mutants. Our data indicate that DsbC can assist DsbA in a DsbD‐independent manner to oxidatively fold envelope proteins. The view that DsbCs function is limited to the disulphide isomerization pathway should therefore be reinterpreted.

Sulfenome mining in Arabidopsis thaliana

Significance When oxygen gets incompletely reduced, reactive oxygen species (ROS) are generated. These ROS molecules can harm the building blocks of the cell but are also important signaling molecules. Until now, the ROS language of the cell has not been understood and a clear view is needed on how the cell differentiates metabolic ROS noise from ROS that allows signaling, regulation, and protection. To address this question, we focused on Arabidopsis thaliana and identified the proteins that react with hydrogen peroxide on the thiol of the amino acid cysteine, which after reaction forms a sulfenic acid. The characterization of the plant sulfenome improves the understanding of important ROS signaling pathways. Reactive oxygen species (ROS) have been shown to be potent signaling molecules. Today, oxidation of cysteine residues is a well-recognized posttranslational protein modification, but the signaling processes steered by such oxidations are poorly understood. To gain insight into the cysteine thiol-dependent ROS signaling in Arabidopsis thaliana, we identified the hydrogen peroxide (H2O2)-dependent sulfenome: that is, proteins with at least one cysteine thiol oxidized to a sulfenic acid. By means of a genetic construct consisting of a fusion between the C-terminal domain of the yeast (Saccharomyces cerevisiae) AP-1–like (YAP1) transcription factor and a tandem affinity purification tag, we detected ∼100 sulfenylated proteins in Arabidopsis cell suspensions exposed to H2O2 stress. The in vivo YAP1-based trapping of sulfenylated proteins was validated by a targeted in vitro analysis of DEHYDROASCORBATE REDUCTASE2 (DHAR2). In DHAR2, the active site nucleophilic cysteine is regulated through a sulfenic acid-dependent switch, leading to S-glutathionylation, a protein modification that protects the protein against oxidative damage.