Biochemical and Biophysical Research Communications
The Escherichia coli btuE gene, encodes a glutathione peroxidase that is induced under oxidative stress conditions
Research highlights
► BtuE is the first bacterial glutathione peroxidase characterized biochemically. ► BtuE is the first glutathione peroxidase described in Escherichia coli. ► BtuE uses GSH, TrxA or TrxC as reducinf agents in vitro. ► btuE transcription is induced under oxidative stress conditions.
Introduction
Aerobic organisms have evolved a number of enzymatic and non-enzymatic antioxidant defense systems which function in a cooperative manner to protect the cell from oxidative stress [1]. Examples of enzymatic antioxidant mechanisms include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). In eukaryotic organisms, GPXs are one of the major enzymatic defenses against hydroperoxides and/or membrane lipid peroxidation [2]. However, little is known about prokaryotic glutathione peroxidases. Exceptions are Neisseria meningitidis and Streptococcus pyogenes, where it has been shown that GPXs are involved in defense against oxidative stress [3], [4], [5], [6]. In general, GPXs catalyze the decomposition of organic hydroperoxides (ROOH) and hydrogen peroxide (H2O2) according to the reaction [7], [8]:ROOH + 2GSH → ROH + GSSG + H2Owhere GSH and GSSG denote reduced and oxidized glutathione, respectively.
The Escherichia coli genome displays an open reading frame exhibiting striking similarity to other genes encoding glutathione peroxidases [9]. This gene, btuE, lies within the btuCED operon, involved in vitamin B12 transport [10], [11]. In spite of its genetic neighborhood, btuE seems not to be involved in B12 transport; in fact, deletions up to 82% of btuE does not affect B12 transport in E. coli[12], which actually depends on E. coli btuC, btuD and btuF genes [13], [14].
To date, available evidence about BtuE function is merely theoretical and suggests that this enzyme, a putative member of the phospholipid glutathione peroxidase family [15], could function as a selenium-independent GPX [16]. The 552 bp E. coli btuE gene encodes a protein of 183 amino acid residues with a Mr of 20 kDa. Like most GPXs, BtuE contains the conserved Cys, Trp and Gln residues at the active site. Although exhibiting a putative oligomerization interface, the lack of the tetramerization “PGGG” motif predicts a homodimeric structure for BtuE [9].
In this work, we characterized biochemically the btuE gene product and found that BtuE catalyzes the decomposition of a variety of peroxides in the presence of thioredoxin A or C as the reducing agent, confirming that its active site confers non-specific peroxidase activity. We also found that btuE expression was induced under oxidative stress conditions and that it is paralleled by an increased BtuE synthesis.
Section snippets
Bacteria and culture conditions
Bacteria were grown routinely in LB medium [17] at 37 °C with shaking. Growth was initiated by inoculating fresh LB medium with 1:100 dilutions of overnight cultures. Solid media contained 2% (w/v) agar and plates were incubated overnight at 37 °C.
The E. coli btuE::lacZ strain was constructed using E. coli BW25113 ΔbtuE (btuE::kan, NARA Institute, Japan) and plasmids pCP20 and pCE37, essentially as described [18]. Integration and correct orientation of pCE37 in the host chromosome was analyzed by
Results and discussion
Although the E. coli BtuE protein was suspected to function as a glutathione peroxidase [15], the experimental evidence was missing. In this context, the aim of this work was to purify BtuE from this bacterium and to characterize its peroxidase activity. BtuE was purified near to homogeneity by affinity chromatography as described in Section 2; BCP peroxidase was purified in parallel and used as positive control for peroxidase activity (Fig. S1). Since BtuE reducing substrates were not known,
Acknowledgments
This work was supported by grants # 1090097 from Fondecyt and Dicyt-USACH, to C.C.V, and from National Institutes of Health # GM049640 to J.A.I. F.A.A. and W.A.D. received doctoral fellowships from Conicyt and from MECESUP-ChileUCH407 and UCH607 (F.A.A.). J.M.P. was sponsored by a postdoctoral fellowship from Conicyt, Chile.
References (31)
- et al.
Construction of targeted single copy lac fusions using l Red and FLP-mediated site-specific recombination in bacteria
Gene
(2002) - et al.
Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth
J. Biol. Chem.
(2004) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Genetic dissection of the phospholipid hydroperoxidase activity of yeast gpx 3 reveals its functional importance
J. Biol. Chem.
(2004) - et al.
The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase
J. Biol. Chem.
(1994) - et al.
YqhD is an aldehyde reductase that protects Escherichia coli from harmful lipid peroxidation-derived aldehydes
J. Biol. Chem.
(2008) - et al.
Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells
J. Biol. Chem.
(2006) Pathways of oxidative damage
Annu. Rev. Microbiol.
(2003)The glutathione peroxidases
Cell. Mol. Life Sci.
(2000)- et al.
Isolation and identification of a glutathione peroxidase homolog gene, gpxA present in Neisseria meningitidis but absent in Neisseria gonorrhoeae
Infect. Immun.
(1995)