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

Abstract

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 thatmore » 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.« less

Authors:
 [1]; ;  [2]; ;  [3];  [1]
  1. School of Biological Sciences Harborne Building, Whiteknights Campus, Reading, RG6 6AS (United Kingdom)
  2. Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS (United Kingdom)
  3. Circular Dichroism Group, Diamond Light Source, Chiltern, Oxfordshire,OX11 0DE (United Kingdom)
Publication Date:
OSTI Identifier:
22202709
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 398; Journal Issue: 3; Other Information: Copyright (c) 2010 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMINO ACIDS; CATIONS; CRYSTALLIZATION; DICHROISM; ESCHERICHIA COLI; IRON; MASS SPECTROSCOPY; MOLECULAR WEIGHT; MONOCRYSTALS; ORTHORHOMBIC LATTICES; PEPTIDES; PSEUDOMONAS

Citation Formats

Rajasekaran, Mohan B, Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS, Mitchell, Sue A, Gibson, Trevor M, Hussain, Rohanah, Siligardi, Giuliano, Andrews, Simon C, Watson, Kimberly A, E-mail: k.a.watson@reading.ac.uk, and Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS. Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae. United States: N. p., 2010. Web. doi:10.1016/J.BBRC.2010.06.072.
Rajasekaran, Mohan B, Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS, Mitchell, Sue A, Gibson, Trevor M, Hussain, Rohanah, Siligardi, Giuliano, Andrews, Simon C, Watson, Kimberly A, E-mail: k.a.watson@reading.ac.uk, & Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS. Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae. United States. doi:10.1016/J.BBRC.2010.06.072.
Rajasekaran, Mohan B, Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS, Mitchell, Sue A, Gibson, Trevor M, Hussain, Rohanah, Siligardi, Giuliano, Andrews, Simon C, Watson, Kimberly A, E-mail: k.a.watson@reading.ac.uk, and Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS. Fri . "Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae". United States. doi:10.1016/J.BBRC.2010.06.072.
@article{osti_22202709,
title = {Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae},
author = {Rajasekaran, Mohan B and Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS and Mitchell, Sue A and Gibson, Trevor M and Hussain, Rohanah and Siligardi, Giuliano and Andrews, Simon C and Watson, Kimberly A, E-mail: k.a.watson@reading.ac.uk and Structural Biology Unit at The BioCentre, University of Reading, Harborne Building, Whiteknights Campus, Reading, RG6 6AS},
abstractNote = {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.},
doi = {10.1016/J.BBRC.2010.06.072},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 3,
volume = 398,
place = {United States},
year = {2010},
month = {7}
}