Abstract
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 approximately 2500 to approximately 8000 ng/mg). Grx3 and Trx2 levels were quite stable during growth ( approximately 4500 and approximately 200 ng/mg, respectively). Grx1 levels decreased from approximately 600 ng/mg at the exponential phase to approximately 285 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.
Highlights
Escherichia coli employs two separate pathways that use NADPH to reduce cytosolic disulfides: the thioredoxin and the glutaredoxin systems
Levels of Thioredoxins 1 and 2 and Glutaredoxins 1, 2, and 3 in a Wild Type Strain—Wild type (DHB4) strain was grown in 1 liter of culture for 5 days in a 3-liter Erlenmeyer flask in LB media (Table III)
General Redoxin Levels—The striking finding is that Grx2, an atypical large glutaredoxin with structure similar to glutathione S-transferase [24, 32], exists in the cell in amounts as high up as 1% of total protein
Summary
Escherichia coli employs two separate pathways that use NADPH to reduce cytosolic disulfides: the thioredoxin and the glutaredoxin systems. In the glutaredoxin system electrons are transferred from NADPH to glutathione reductase (GR), to glutathione (GSH), and to glutaredoxins 1, 2, and 3 (Grx, Grx, and Grx3) Thioredoxins reduce their substrates by employing a dithiol mechanism provided by an active site of two redoxactive cysteines separated by two other amino acids (e.g. CXXC). All glutaredoxins are good in vitro electron donors for the reduction of arsenate by arsenate reductase (ArsC) [9], with Grx being 100-fold more efficient than Grx1 [10] In addition to their specific enzyme-linked electron donor activities, Trx, Trx, and to a lesser extent, Grx and Grx are involved in the general reduction of cytosolic disulfides as envisaged from experiments examining the folding of leaderless alkaline phosphatase in the E. coli cytosol [8, 11]. In this study we have determined the actual protein levels of the two
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