Interaction domain on thioredoxin for Pseudomonas aeruginosa 5′-adenylylsulfate reductase

Jung Sung Chung, Valérie Noguera-Mazon, Jean Marc Lancellin, Sung Kun Kim, Masakazu Hirasawa, Maggy Hologne, Thomas Leustek, David B. Knaff

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

NMR spectroscopy has been used to map the interaction domain on Escherichia coli thioredoxin for the thioredoxin-dependent 5′-adenylylsulfate reductase from Pseudomonas aeruginosa (PaAPR). Seventeen thioredoxin amino acids, all clustered around Cys-32 (the more surface-exposed of the two active-site cysteines), have been located at the PaAPR binding site. The center of the binding domain is dominated by nonpolar amino acids, with a smaller number of charged and polar amino acids located on the periphery of the site. Twelve of the amino acids detected by NMR have non-polar, hydrophobic side chains, including one aromatic amino acid (Trp-31). Four of the thioredoxin amino acids at the PaAPR binding site have polar side chains (Lys-36, Asp-61, Gln-62 and Arg-73), with three of the four having charged side chains. Site-directed mutagenesis experiments have shown that replacement of Lys-36, Asp-61, and Arg-73 and of the absolutely conserved Trp-31 significantly decreases the Vmax for the PaAPR-catalyzed reduction of 5′-adenylylsulfate, with E. coli thioredoxin serving as the electron donor. The most dramatic effect was observed with the W31A variant, which showed no activity as a donor to PaAPR. Although the thiol of the active-site Cys-256 of PaAPR is the point of the initial nucleophilic attack by reduced thioredoxin, mutagenic replacement of Cys-256 by serine has no effect on thioredoxin binding to PaAPR.

Original languageEnglish (US)
Pages (from-to)31181-31189
Number of pages9
JournalJournal of Biological Chemistry
Volume284
Issue number45
DOIs
StatePublished - Nov 6 2009

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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