Interaction Domain on Thioredoxin for Pseudomonas aeruginosa 5′-Adenylylsulfate Reductase*

AUTOR(ES)

Chung, Jung-Sung

FONTE

American Society for Biochemistry and Molecular Biology

RESUMO

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.

Documentos Relacionados

• Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5′-adenylylsulfate reductase
• Sulfate reduction in higher plants: Molecular evidence for a novel 5′-adenylylsulfate reductase
• Characterization of Sulfate Assimilation in Marine Algae Focusing on the Enzyme 5′-Adenylylsulfate Reductase1
• Identification of a New Class of 5′-Adenylylsulfate (APS) Reductases from Sulfate-Assimilating Bacteria
• Differential Subcellular Localization and Expression of ATP Sulfurylase and 5′-Adenylylsulfate Reductase during Ontogenesis of Arabidopsis Leaves Indicates That Cytosolic and Plastid Forms of ATP Sulfurylase May Have Specialized Functions1