The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion

• Published in: Microbiology (Society for General Microbiology) 2002-08, Vol.148 (8), p.2449-2456
• Main Authors: Lewin, Allison C, Doughty, Phillip A, Flegg, Lynda, Moore, Geoffrey R, Spiro, Stephen
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Binding Sites; Biological and medical sciences; Carrier Proteins; Circular Dichroism; Cysteine - chemistry; Cysteine - metabolism; Escherichia coli - chemistry; Escherichia coli - genetics; Ferric Compounds - chemistry; Fundamental and applied biological sciences. Psychology; fur protein; Genetics; Histidine - metabolism; Maltose-binding protein; Maltose-Binding Proteins; Metabolism. Enzymes; Microbiology; Protein Conformation; Pseudomonas aeruginosa; Pseudomonas aeruginosa - chemistry; Pseudomonas aeruginosa - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Repressor Proteins - metabolism; Spectrometry, Fluorescence; Zinc - chemistry; Zinc - metabolism
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-148-8-2449

School of Biological Sciences 1 and School of Chemical Sciences 2 , University of East Anglia, Norwich NR4 7TJ, UK Author for correspondence: Stephen Spiro. Tel: [phone]. Fax: [phone]. e-mail: s.spiro{at}uea.ac.uk The ferric uptake regulator (Fur) of Pseudomonas aeruginosa was expressed in Escherichia coli in its native form and as a fusion to the maltose-binding protein (MBP). Fur from the MBP fusion bound to MBP after proteolytic cleavage, and the two could only be separated by partial unfolding. The refolded protein was in the same conformation as native protein (as judged by circular dichroism and fluorescence spectroscopies) and was fully active in DNA-binding assays. As-prepared native Fur contained small amounts of Zn 2+ that were easily removed by treatment with EDTA, and apo-protein could be reconstituted with approximately one Zn 2+ ion per monomer. Thus, the P. aeruginosa Fur can probably accommodate a single Zn 2+ ion bound to the metal-sensing site. The single cysteine residue of P. aeruginosa Fur aligns with a cysteine in other members of the Fur family that is essential for activity of the E. coli protein, and is believed to provide one of the ligands to a structural Zn 2+ ion. This cysteine residue was shown to be dispensable for the in vivo activity of P. aeruginosa Fur, which is consistent with the suggestion that the P. aeruginosa protein does not contain a structural Zn 2+ ion. Members of the Fur family contain a highly conserved His-His-Asp-His motif. Alanine substitutions of residues in this motif showed His-87 and His-89 of P. aeruginosa Fur to be essential for activity, whilst His-86 and Asp-88 are partially dispensable. Keywords: Fur, iron regulation Abbreviations: CD, circular dichroism; Gdn . HCl, guanidinium hydrochloride; MBP, maltose-binding protein a Present address: School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.