Disulfide Bond Formation and Activation of Escherichia coli β-Galactosidase under Oxidizing Conditions

Abstract

<jats:title>ABSTRACT</jats:title><jats:p><jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>β-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version of<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>β-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation.</jats:p>

Synonymous Codons Direct Cotranslational Folding toward Different Protein Conformations

“…It cannot be excluded that these cysteines may become oxidized in cytosol of E. coli . Although disulfide bonds are rarely formed in E. coli cytoplasmic proteins, exceptions to this general rule do exist (Aslund et al, 1999; Jakob et al, 1999; Seras-Franzoso et al, 2012). Our NMR analysis of U, H-P1 and H-P2 revealed chemical shift differences in particular for the six cysteine residues at positions 15, 18, 22, 32, 41, and 78 (in NTD) (Figure 2C; inset and Table 1), both in 1D spectra (Figure 2D) and in 2D- 1 H- 15 N correlated experiments (Figure 2E and 2F).…”

Engineering the genetic components of a whole‐cell catalyst for improved enzymatic CO2 capture and utilization

“…Correct formation of disulfide bonds on an unfolded protein followed by the correct folding of the protein is catalyzed in the periplasm by the Dsb machinery (Berkmen, ). Thus, the higher free thiol content of SP TorA :: hm CA is indicative of a higher fraction of (partially) misfolded enzymes, resulting in lower specific activity (Seras‐Franzoso et al, ). In addition, the purified SP PelB :: hm CA showed a higher thermal stability with a half‐life of 7.6 hr compared with that of SP TorA :: hm CA with a half‐life of 5.6 hr at 60°C (Figure d).…”

Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens

“…A recent study suggested that this lack of activity was due to the presence of the glutathione system in Gram-negative bacteria which helps to mediate resistance to auranofin in these pathogens 13 . However, when we assessed auranofin’s antibacterial activity against both wild-type and Origami-2 ( trxb / gor double mutant) E. coli mutant strains, neither strain was susceptible to auranofin even at a concentration of 16 μg/ml 40 . This suggests an alternative mechanism may be responsible for the lack of activity observed with auranofin against Gram-negative bacteria.…”