Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae
- School of Biological Sciences Harborne Building, Whiteknights Campus, Reading, RG6 6AS (United Kingdom)
- Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS (United Kingdom)
- Circular Dichroism Group, Diamond Light Source, Chiltern, Oxfordshire,OX11 0DE (United Kingdom)
Research highlights: {yields} Bioinformatic analysis reveals EfeM is a metallopeptidase with conserved HXXE motif. {yields} Mass spectrometry confirms EfeM consists of 251 residues, molecular weight 27,772Da. {yields} SRCD spectroscopy shows an {alpha}-helical secondary structure. {yields} Single crystals of EfeM are orthorhombic and diffract to 1.6A resolution. {yields} Space group is P22{sub 1}2{sub 1} with cell dimensions a = 46.74, b = 95.17 and c = 152.61 A. -- Abstract: The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal 'Peptidase{sub M}75' domain of 251 residues. The C-terminal domain contains a highly conserved 'HXXE' motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or 'Psyr{sub 3}370') encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M{sub W} 27,772 Da). Circular dichroism spectroscopy of EfeM indicated a mainly {alpha}-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6 A. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.
- OSTI ID:
- 22202709
- Journal Information:
- Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 3 Vol. 398; ISSN BBRCA9; ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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