The structure of a triple mutant of pI258 arsenate reductase from Staphylococcus aureus and its 5-thio-2-nitrobenzoic acid adduct.

TitleThe structure of a triple mutant of pI258 arsenate reductase from Staphylococcus aureus and its 5-thio-2-nitrobenzoic acid adduct.
Publication TypeJournal Article
Year of Publication2004
AuthorsMessens, J., I. Van Molle, P. Vanhaesebrouck, K. Van Belle, K. Wahni, J. C. Martins, L. Wyns, and R. Loris
JournalActa Crystallogr D Biol Crystallogr
Volume60
IssuePt 6
Pagination1180-4
Date Published2004 Jun
ISSN0907-4449
KeywordsArsenite Transporting ATPases, Crystallography, X-Ray, Dimerization, Disulfides, Electrons, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Histidine, Ion Pumps, Mass Spectrometry, Models, Molecular, Multienzyme Complexes, Mutagenesis, Site-Directed, Mutation, Nitrobenzoates, Oxidation-Reduction, Protein Conformation, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Staphylococcus aureus, Sulfhydryl Compounds
Abstract

Structural insights into formation of the complex between the ubiquitous thiol-disulfide oxidoreductase thioredoxin and its oxidized substrate are under-documented owing to its entropical instability. In vitro, it is possible via a reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) to make a stable mixed-disulfide complex between thioredoxin from Staphylococcus aureus and one of its substrates, oxidized pI258 arsenate reductase (ArsC) from S. aureus. In the absence of the crystal structure of an ArsC-thioredoxin complex, the structures of two precursors of the complex, the ArsC triple mutant ArsC C10SC15AC82S and its 5-thio-2-nitrobenzoic acid (TNB) adduct, were determined. The ArsC triple mutant has a structure very similar to that of the reduced form of wild-type ArsC, with a folded redox helix and a buried catalytic Cys89. In the adduct form, the TNB molecule is buried in a hydrophobic pocket and the disulfide bridge between TNB and Cys89 is sterically inaccessible to thioredoxin. In order to form a mixed disulfide between ArsC and thioredoxin, a change in the orientation of the TNB-Cys89 disulfide in the structure is necessary.

DOI10.1107/S0907444904007334
Alternate JournalActa Crystallogr. D Biol. Crystallogr.
PubMed ID15159594
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