The Escherichia coli btuE gene, encodes a glutathione peroxidase that is induced under oxidative stress conditions

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Abstract

Most aerobic organisms are exposed to oxidative stress. Looking for enzyme activities involved in the bacterial response to this kind of stress, we focused on the btuE-encoded Escherichia coli BtuE, an enzyme that shares homology with the glutathione peroxidase (GPX) family. This work deals with the purification and characterization of the btuE gene product.

Purified BtuE decomposes in vitro hydrogen peroxide in a glutathione-dependent manner. BtuE also utilizes preferentially thioredoxin A to decompose hydrogen peroxide as well as cumene-, tert-butyl-, and linoleic acid hydroperoxides, confirming that its active site confers non-specific peroxidase activity. These data suggest that the enzyme may have one or more organic hydroperoxide as its physiological substrate.

The btuE gene was induced when cells were exposed to oxidative stress elicitors that included potassium tellurite, menadione and hydrogen peroxide, among others, suggesting that BtuE could participate in the E. coli response to reactive oxygen species. To our knowledge, this is the first report describing a glutathione peroxidase in E. coli.

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.

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