Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

Rajasekaran, M. B., Mitchell, S. A., Gibson, T. M., Hussain, R., Siligardi, G., Andrews, S. C. ORCID: https://orcid.org/0000-0003-4295-2686 and Watson, K. A. ORCID: https://orcid.org/0000-0002-9987-8539 (2010) Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae. Biochemical and Biophysical Research Communications, 398 (3). pp. 366-371. ISSN 0006-291X doi: 10.1016/j.bbrc.2010.06.072

Abstract/Summary

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 (M(W) 27,772Da). 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 1.6A. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.