Interaction domain on thioredoxin for Pseudomonas aeruginosa 5'-adenylylsulfate reductase.
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 V(max) 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.
Chung
Jung-Sung
JS
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
Noguera-Mazon
Valérie
V
Lancelin
Jean-Marc
JM
Kim
Sung-Kun
SK
Hirasawa
Masakazu
M
Hologne
Maggy
M
Leustek
Thomas
T
Knaff
David B
DB
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D013879:Thioredoxins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase