Transcriptional Regulation of Glutaredoxin and Thioredoxin Pathways and Related Enzymes in Response to Oxidative Stress

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108

Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys 95 , Cys 82 , and Cys 61 , and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Oxidative stress defenses combat reactive oxygen species (1, 2) such as superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical (OH ⅐ ) generated by the host immune response, environmental factors, and the incomplete reduction of oxygen to water during aerobic respiration, all of which are hazardous to proteins, DNA, and lipids (3, 4). Escherichia coli protects cellular components from oxidative damage by employing a variety of antioxidant defense enzymes, such as hydroperoxidases (catalases) I and II (gene products of katG and katE, respectively) that decompose H 2 O 2 (5), and superoxide dismutases (manganese superoxide dismutase, sodA; iron superoxide dismutase, sodB; copper-zinc superoxide dismutase, sodC) (6) that eliminate O 2 . . Additional defenses in E. coli against alkyl and lipid hydroperoxides are provided by multiple, non-heme peroxidases including 1) the peroxidatic component from the alkyl hydroperoxide reductase system (AhpC) 1 (7); 2) a weakly active, thioredoxin (Trx)-dependent bacterioferritin-comigratory protein (BCP) (8); and 3) the periplasmic thiol peroxidase (Tpx, p20, scavengase) (9). In addition, a glutathione peroxidase homologue, the gene product of btuE (10), has been identified in E. coli, and preliminary investigations have indicated Trx-dependent peroxidatic activity against organic hydroperoxides (ROOH) and H 2 O 2 .2

Section: Characterization Of Purified Tpx and Mutants-homogene-mentioning

confidence: 99%

Catalytic Mechanism of Thiol Peroxidase from Escherichia coli

Baker

Poole

2003

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108

“…Previous studies have reported the regulation of thioredoxins and glutaredoxins at their transcriptional level (14,18). In this study we have determined the actual protein levels of the two thioredoxins and the three glutaredoxins by ELISA.…”

mentioning

confidence: 99%

“…The transcription factor OxyR regulates the transcription of GR, Grx1, and Trx2 under oxidative conditions (15)(16)(17). grxA mRNA levels are highly increased in response to oxidative stress (18). Grx1 and Trx1 are able to reduce and thus deactivate OxyR in vitro, but Grx1 seems to be the preferred reductant in vivo (19,20).…”

mentioning

confidence: 99%

Protein Levels of Escherichia coli Thioredoxins and Glutaredoxins and Their Relation to Null Mutants, Growth Phase, and Function

Potamitou

Holmgren

Vlamis-Gardikas

2002

Self Cite

Levels of Escherichia coli thioredoxin 1 (Trx1), Trx2, glutaredoxin 1 (Grx1), Grx2, and Grx3 have been determined by novel sensitive sandwich enzyme-linked immunosorbent assay. In a wild type strain, levels of Trx1 increased from the exponential to the stationary phase of growth (1.5-fold to 3400 ng/mg), as did levels of Grx2 (from ϳ2500 to ϳ8000 ng/mg). Grx3 and Trx2 levels were quite stable during growth (ϳ4500 and ϳ200 ng/mg, respectively). Grx1 levels decreased from ϳ600 ng/mg at the exponential phase to ϳ285 ng/mg at the stationary phase. A large elevation of Grx1 (20 -30-fold), was observed in null mutants for the thioredoxin system whereas levels of the other redoxins in all combinations of examined null mutants barely exceeded a 2-3-fold increase. Measurements of thymidine incorporation in newly synthesized DNA suggested that mainly Grx1 and, to a lesser extent, Trx1 contribute to the reduction of ribonucleotides. All glutaredoxin species were elevated in catalase-deficient strains, implying an antioxidant role for the glutaredoxins. Trx1, Trx2, and Grx1 levels increased after exposure to hydrogen peroxide and decreased after exposure to mercaptoethanol. The levels of Grx2 and Grx3 behaved exactly the opposite, suggesting that the transcription factor OxyR does not regulate their expression.Escherichia coli employs two separate pathways that use NADPH to reduce cytosolic disulfides: the thioredoxin and the glutaredoxin systems. The thioredoxin system consists of thioredoxin reductase and thioredoxins 1 and 2 (Trx1 and Trx2).

“…Regulation of nrdAB expression in E. coli has been shown to be very complex; multiple cis-acting positive regulatory sites identified upstream of the nrdAB promoter appear to control, independently but in concert, the cell cycle-dependent transcription and the response to inhibition of DNA synthesis (9). Recent data from our group add further complexity to the nrdAB regulation, demonstrating a tight and inverse relation between expression of nrdAB and that of genes coding for components of both glutaredoxin and thioredoxin pathways (10). Interestingly, we have observed that induction ratios of nrdAB transcription by hydroxyurea (RRase inhibitor) are similar to the increments in nrdAB basal expression of mutants lacking both Trx1 and Grx1.…”

mentioning

confidence: 99%

“…We have quantitated the in vivo transcription of genes encoding for components of both glutaredoxin and thioredoxin pathways by means of a novel multiplex reverse transcriptionpolymerase chain reaction (RT-PCR) approach (10,12). In this protocol, all target genes, a housekeeping gene (also named control gene, reference gene, or internal standard), and one external standard are amplified in the same reaction tube.…”

mentioning

confidence: 99%

Expression Analysis of the nrdHIEF Operon fromEscherichia coli

Monje-Casas

Jurado

Prieto‐Álamo

et al. 2001

Self Cite

Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB and nrdHIEF operons. While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function. Here, we present evidence that nrdHIEF expression might be important under certain circumstances. Basal transcript levels were dramatically enhanced (25-75-fold), depending on the growth-phase and the growthmedium composition. Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants. Moreover, nrdHIEF expression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium). The mechanism(s) that triggers nrdHIEF expression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of the nrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. coli might optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors.