Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade

Abstract

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122–Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122–Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs

“…Mass spectometry analysis was performed as described previously 54 , 55 . After immunoprecipitation, samples were resolved using a 10% Tris–Glycine SDS-PAGE and proteins were visualized using PageBlue (Thermo Scientific, 24,620).…”

Identification and Quantification of S-Sulfenylation Proteome of Mycobacterium tuberculosis under Oxidative Stress

“…MsrB (I6YA00), Thiol peroxidase (Tpx, P9WG35 ), and Alkyl hydroperoxide reductase C (AhpC, P9WQB7 ) have been reported as the key elements in the defense against oxidative stress in Mtb ( 24 , 35 , 36 ). And the cysteine of Thiol peroxidase from Escherichia coli and MsrB from C. diphtheriae could be oxidized to sulfuric acid (SOH) by peroxide ( 37 , 38 ). Interestingly, elongation factors were reported as targets of oxidation by ROS via the oxidation of cysteine residues to SOH ( 39 ).…”

Profiling the Site of Protein CoAlation and Coenzyme A Stabilization Interactions

“…Our study showed that residues within the CoAlated cysteine environment contribute to the stabilization of both CoA and the CoAlated cysteine, facilitating CoAlation. Similar to CoA, glutathione (GSH— Figure 4 A), mycothiol and bacillithiol are low molecular weight (LMW) thiols, which protect protein cysteines from hyperoxidation and are implicated in redox signaling [ 19 , 27 , 47 , 48 ]. Although research on the detailed site(s) of mycothiolation/bacillithiolation is not thoroughly discussed, protein glutathionylation on the other hand, is well studied [ 49 ].…”

“…When sulfenic acid is formed on a cysteine, LMW thiols (GSH, BSH, MSH and CoA) can perform a nucleophilic attack on the sulfur atom of the sulfenic acid and form a mixed-disulfide bond in a process termed thiolation (glutathionylation, bacillithiolation, mycothiolation and CoAlation). This prevents the hyperoxidation of the protein cysteine and in many cases, its irreversible loss of function [ 8 , 10 , 18 , 19 , 20 , 21 ] ( Figure 1 B). Protein thiolation could also occur through thiol-disulfide exchange reactions.…”