Characterization of Escherichia coli Null Mutants for Glutaredoxin 2

Abstract

Three Escherichia coli glutaredoxins catalyze GSHdisulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB ؊ ) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB ؊ cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.

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

“…1), with Grx2 reaching Ͼ20 g/mg in the katEkatGgshA minus strain. These data correlate well with the reported up-regulation of total glutaredoxin activity in the same strains (8).…”

“…Grx1 (encoded by grxA) can also reduce RR1a and PAPS reductase (6). Combined null mutants for trxA and grxA provided evidence for Grx2 (encoded by grxB) and Grx3 (encoded by grxC), which contributes to more than 80% of total GSH oxidoreductase activity using ␤-hydroxyetyl disulfide as substrate (7,8). Grx3 is a poor in vitro electron donor for RR1a (about 5% of the catalytic activity of Grx1), and Grx2 lacks such activity altogether.…”

“…In vitro experiments have shown that Grx1 can reduce the disulfide of PAPS reductase, whereas Grx2 and Grx3 were not active in this assay (4). Grx2 protects intracellular proteins from carbonylation damage occurring after exposure to hydrogen (8). All glutaredoxins are good in vitro electron donors for the reduction of arsenate by arsenate reductase (ArsC) (9), with Grx2 being 100-fold more efficient than Grx1 (10).…”

“…All glutaredoxins are good in vitro electron donors for the reduction of arsenate by arsenate reductase (ArsC) (9), with Grx2 being 100-fold more efficient than Grx1 (10). In addition to their specific enzyme-linked electron donor activities, Trx1, Trx2, and to a lesser extent, Grx1 and Grx2 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).…”

Oxidation and S-Nitrosylation of Cysteines in Human Cytosolic and Mitochondrial Glutaredoxins

“…Grxs are involved in many different cellular processes, e.g. DNA synthesis (10), sulfur assimilation (11,12), defense against oxidative stress (13)(14)(15), apoptosis (16), cellular differentiation (17), regulation of transcription factor binding activity (18), and redox regulation via reactive oxygen species.…”