Characterization and function of Escherichia coli glutaredoxins

Abstract

Escherichia coli employs two separate pathways, driven by NADPH to reduce protein disulfides: the thioredoxin and glutaredoxin systems. Both systems function via redox active disulfides and are involved in many cellular functions including, the synthesis of DNA building blocks (by reducing the essential enzyme ribonucleotide reductase), the generation of reduced sulfur (via PAPS reductase), and the repair of oxidative damage to protein (by methionine sulfoxide reductase).

This work aims in a better understanding of the two systems with emphasis on glutaredoxins. Sensitive ELISAs for the two thioredoxins (Trx1, Trx2) and the three glutaredoxins (Grx1, Grx2, Grx3) of E. coli were developed, and protein levels measured at different stages of growth and in different genetic backgrounds.

We found that glutaredoxins, were involved in antioxidant defense. Levels of all three glutaredoxins were elevated in catalase deficient strains, particularly when combined with null mutants for the thioredoxin or glutaredoxin systems. OxyR did not affect the levels of Grx2 or Grx3, as it does for Grx1, instead Grx2 levels were elevated in an oxyR null mutant. Grx1 and Grx2 contributed to the defense against protein carbonylation damage caused by hydrogen peroxide.

Measurements of thymidine incorporation in newly synthesized DNA in relevant null mutants, showed that it is mainly Grx 1 and to a lesser extent Trx 1 that are involved in the reduction of deoxyribonucleotides. Grx2 was the most abundant glutaredoxin, with levels increasing at the stationary phase of growth up to one per cent of total soluble protein. Guanosine-3',5'-tetraphoshate (ppGpp) and sigma-s that regulate the transcription of genes in the stationary phase of growth, affected dramatically the expression of Grx2, as did osmotic pressure and cAMP, presumably via a.

In accordance with the role of Grx2 as a stationary phase protein, null mutants for grxB were lysing at the stationary phase of growth and exhibited a distorted morphology. Null mutants for grxB and all three glutaredoxin genes were viable in rich and minimal media. However, a combined null mutant for all three glutaredoxins and glutathione reductase (gor-grxA-grxB-grxC) was barely growing on minimal media, suggesting the possibility of a mixed disulfide mechanism for the regulation of the activity of PAPS reductase. In fact, a glutathionylated species was detected in vivo in poorly growing gor-grxA-grxB-grxC.

In vitro incubation of PAPS reductase with oxidized glutathione lead to the enzyme's inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys239. This species could be reduced and its activity restored by glutaredoxins. Reversible glutathionylation may thus regulate the activity of PAPS reductase. A novel highly abundant monothiol glutaredoxin (Grx4) was identified with maximum levels at the stationary phase of growth (750-2000 ng/mg). Expression of Grx4 is likely to be regulated by ppGpp, but not sigma-s.