Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae |
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Authors: | Mohan B Rajasekaran Sue A Mitchell Rohanah Hussain Simon C Andrews |
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Affiliation: | a School of Biological Sciences Harborne Building, Whiteknights Campus, Reading, RG6 6AS, UK b Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS, UK c Circular Dichroism Group, Diamond Light Source, Chiltern, Oxfordshire,OX11 0DE, UK |
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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_M75’ 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_3370’) 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 (MW 27,772 Da). Circular dichroism spectroscopy of EfeM indicated a mainly α-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 Å. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role. |
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Keywords: | Iron transport Peptidase_M75 SRCD Mass spectrometry Crystallisation EfeUOB Psyr_3370 |
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