Sulfur Denitrosylation by an Engineered Trx-like DsbG Enzyme Identifies Nucleophilic Cysteine Hydrogen Bonds as Key Functional Determinant

Abstract

Exposure of bacteria to NO results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as GSH. The cells possess enzymatic systems that catalyze the denitrosylation of these modified sulfurs. An important player in these systems is thioredoxin (Trx), a ubiquitous, cytoplasmic oxidoreductase that can denitrosylate proteins in vivo and S-nitrosoglutathione (GSNO) in vitro. However, a periplasmic or extracellular denitrosylase has not been identified, raising the question of how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested whether DsbG and DsbC, two Trx family proteins that function in reducing pathways in the Escherichia coli periplasm, also possess denitrosylating activity. Both DsbG and DsbC are poorly reactive toward GSNO. Moreover, DsbG is unable to denitrosylate its specific substrate protein, YbiS. Remarkably, by borrowing the CGPC active site of E. coli Trx-1 in combination with a T200M point mutation, we transformed DsbG into an enzyme highly reactive toward GSNO and YbiS. The pK a of the nucleophilic cysteine, as well as the redox and thermodynamic properties of the engineered DsbG are dramatically changed and become similar to those of E. coli Trx-1. X-ray structural insights suggest that this results from a loss of two direct hydrogen bonds to the nucleophilic cysteine sulfur in the DsbG mutant. Our results highlight the plasticity of the Trx structural fold and reveal that the subtle change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds.

The multiplicity of thioredoxin systems meets the specific lifestyles of Clostridia

“…Protein oxidation occurs mainly on cysteine residues, causing the formation of disulfide bonds, of sulfenic (R-SOH) or sulfinic (R-SO 2 H) acids but also the irreversible formation of sulfonic acid (R-SO 3 H). In addition, the presence of nitric oxide (NO) or reactive nitrogen species can cause S-nitrosylation of cysteine [ 2 , 5 , 6 ]. These modifications can inactivate proteins, and cells thus require active repair strategies that include thioredoxin (Trx) systems.…”

“…(B-C) Expression of the trxB1 gene and the CD3605.1 gene encoding a ferredoxin was monitored by qRT-PCR in (B) WT strain after 24 h of growth in TY medium in anaerobiosis or at 1% O2 or in (C) WT strain and sigB mutant after 4.5 h of growth in TY. Experiments were performed in at least 6 Comparison of the C. difficile core genome and trxB4 evolution. The tanglegram representing the trxB4 tree (right) and the core-genome tree pruned to contain only genomes present in the trxB4 tree (left).…”

The multiplicity of Thioredoxin systems meets the specific needs of Clostridia

“…Protein oxidation occurs mainly on cysteine residues, causing the formation of disulfide bonds, of sulfenic (R-SOH) or sulfinic (R-SO 2 H) acids but also the irreversible formation of sulfonic acid (R-SO 3 H). In addition, the presence of nitric oxide (NO) or reactive nitrogen species can cause S-nitrosylation of cysteine [2,5,6]. These modifications can inactivate proteins, and cells thus require active repair strategies that includes thioredoxin (Trx) systems.…”