5g1k
A triple mutant of DsbG engineered for denitrosylationA triple mutant of DsbG engineered for denitrosylation
Structural highlights
FunctionDSBG_ECOLI Involved in disulfide bond formation. DsbG and DsbC are part of a periplasmic reducing system that controls the level of cysteine sulfenylation, and provides reducing equivalents to rescue oxidatively damaged secreted proteins such as ErfK, YbiS and YnhG. Probably also functions as a disulfide isomerase with a narrower substrate specificity than DsbC. DsbG is maintained in a reduced state by DsbD. Displays chaperone activity in both redox states in vitro.[1] Publication Abstract from PubMedExposure of bacteria to nitric oxide (NO) results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as glutathione (GSH). 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 as to how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested if 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 towards 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 towards GSNO and YbiS. The pKa 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 towards nitrosylated compounds. Sulfur denitrosylation by an engineered Trx-like DsbG enzyme identifies nucleophilic cysteine hydrogen bonds as key functional determinant.,Lafaye C, Van Molle I, Tamu Dufe V, Wahni K, Boudier A, Leroy P, Collet JF, Messens J J Biol Chem. 2016 May 18. pii: jbc.M116.729426. PMID:27226614[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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