title: 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 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 toward nitrosylated compounds.
authors.affiliation: From the de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium, WELBIO, B-1200 Brussels, Belgium, the Brussels Center for Redox Biology, B-1050 Brussels, Belgium.
authors.forename: Céline
authors.initials: C
authors.lastname: Lafaye
authors.affiliation: the Brussels Center for Redox Biology, B-1050 Brussels, Belgium, the Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, B-1050 Brussels, Belgium, the Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium, and.
authors.forename: Inge
authors.initials: I
authors.lastname: Van Molle
authors.affiliation: From the de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium, WELBIO, B-1200 Brussels, Belgium, the Brussels Center for Redox Biology, B-1050 Brussels, Belgium, the Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, B-1050 Brussels, Belgium, the Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium, and.
authors.forename: Veronica
authors.initials: V
authors.lastname: Tamu Dufe
authors.affiliation: the Brussels Center for Redox Biology, B-1050 Brussels, Belgium, the Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, B-1050 Brussels, Belgium, the Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium, and.
authors.forename: Khadija
authors.initials: K
authors.lastname: Wahni
authors.affiliation: the Université de Lorraine, Faculté de Pharmacie, 54000 Nancy, France.
authors.forename: Ariane
authors.initials: A
authors.lastname: Boudier
authors.affiliation: the Université de Lorraine, Faculté de Pharmacie, 54000 Nancy, France.
authors.forename: Pierre
authors.identifier: 0000-0002-9389-2698
authors.initials: P
authors.lastname: Leroy
authors.affiliation: From the de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium, WELBIO, B-1200 Brussels, Belgium, the Brussels Center for Redox Biology, B-1050 Brussels, Belgium, jfcollet@uclouvain.be.
authors.forename: Jean-François
authors.identifier: 0000-0001-8069-7036
authors.initials: JF
authors.lastname: Collet
authors.affiliation: the Brussels Center for Redox Biology, B-1050 Brussels, Belgium, the Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, B-1050 Brussels, Belgium, the Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium, and joris.messens@vib-vub.be.
authors.forename: Joris
authors.identifier: 0000-0002-2128-8264
authors.initials: J
authors.lastname: Messens
mesh_terms: D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015538:Nitrosation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; C545765:YbiS protein, E coli; D013879:Thioredoxins; D026422:S-Nitrosoglutathione; D013455:Sulfur; D010088:Oxidoreductases; C109831:DsbG protein, E coli; D003545:Cysteine
keywords: S-nitrosylation; antioxidant; bacteria; bioengineering; crystal structure; reactive nitrogen species (RNS); redox regulation; thiol; thioredoxin