Subcellular localization and in vivo oxidation-reduction kinetics of thiol peroxidase in Escherichia coli.
ABSTRACT Peroxiredoxins are a class of peroxide-scavenging enzymes having a conserved cysteine residue(s) in their active centers. Thiol peroxidase (Tpx) is one of the peroxiredoxins identified in Escherichia coli. Despite the absence of the N-terminal signal sequence for transport across the membrane, it has been characterized as a periplasmic protein. Reanalysis of Tpx localization, using active site cysteine mutants of thioredoxin 1 (Trx1), demonstrated that Tpx forms a mixed-disulfide complex with cytoplasmic Trx1, indicating that Tpx localizes in the cytoplasm.
Article: Structural characterisation of Tpx from Yersinia pseudotuberculosis reveals insights into the binding of salicylidene acylhydrazide compounds.[show abstract] [hide abstract]
ABSTRACT: Thiol peroxidase, Tpx, has been shown to be a target protein of the salicylidene acylhydrazide class of antivirulence compounds. In this study we present the crystal structures of Tpx from Y. pseudotuberculosis (ypTpx) in the oxidised and reduced states, together with the structure of the C61S mutant. The structures solved are consistent with previously solved atypical 2-Cys thiol peroxidases, including that for "forced" reduced states using the C61S mutant. In addition, by investigating the solution structure of ypTpx using small angle X-ray scattering (SAXS), we have confirmed that reduced state ypTpx in solution is a homodimer. The solution structure also reveals flexibility around the dimer interface. Notably, the conformational changes observed between the redox states at the catalytic triad and at the dimer interface have implications for substrate and inhibitor binding. The structural data were used to model the binding of two salicylidene acylhydrazide compounds to the oxidised structure of ypTpx. Overall, the study provides insights into the binding of the salicylidene acylhydrazides to ypTpx, aiding our long-term strategy to understand the mode of action of this class of compounds.PLoS ONE 01/2012; 7(2):e32217. · 4.09 Impact Factor
Article: A new family of membrane electron transporters and its substrates, including a new cell envelope peroxiredoxin, reveal a broadened reductive capacity of the oxidative bacterial cell envelope.[show abstract] [hide abstract]
ABSTRACT: The Escherichia coli membrane protein DsbD functions as an electron hub that dispatches electrons received from the cytoplasmic thioredoxin system to periplasmic oxidoreductases involved in protein disulfide isomerization, cytochrome c biogenesis, and sulfenic acid reduction. Here, we describe a new class of DsbD proteins, named ScsB, whose members are found in proteobacteria and Chlamydia. ScsB has a domain organization similar to that of DsbD, but its amino-terminal domain differs significantly. In DsbD, this domain directly interacts with substrates to reduce them, which suggests that ScsB acts on a different array of substrates. Using Caulobacter crescentus as a model organism, we searched for the substrates of ScsB. We discovered that ScsB provides electrons to the first peroxide reduction pathway identified in the bacterial cell envelope. The reduction pathway comprises a thioredoxin-like protein, TlpA, and a peroxiredoxin, PprX. We show that PprX is a thiol-dependent peroxidase that efficiently reduces both hydrogen peroxide and organic peroxides. Moreover, we identified two additional proteins that depend on ScsB for reduction, a peroxiredoxin-like protein, PrxL, and a novel protein disulfide isomerase, ScsC. Altogether, our results reveal that the array of proteins involved in reductive pathways in the oxidative cell envelope is significantly broader than was previously thought. Moreover, the identification of a new periplasmic peroxiredoxin indicates that in some bacteria, it is important to directly scavenge peroxides in the cell envelope even before they reach the cytoplasm. IMPORTANCE: Peroxides are reactive oxygen species (ROS) that damage cellular components such as lipids, proteins, and nucleic acids. The presence of protection mechanisms against ROS is essential for cell survival. Bacteria express cytoplasmic catalases and thiol-dependent peroxidases to directly scavenge harmful peroxides. We report the identification of a peroxide reduction pathway active in the periplasm of Caulobacter crescentus, which reveals that, in some bacteria, it is important to directly scavenge peroxides in the cell envelope even before they reach the cytoplasm. The electrons required for peroxide reduction are delivered to this pathway by ScsB, a new type of membrane electron transporter. We also identified two additional likely ScsB substrates, including a novel protein disulfide isomerase. Our results reveal that the array of proteins involved in reductive pathways in the oxidative environment of the cell envelope is significantly broader than was previously thought.mBio 01/2012; 3(2). · 5.31 Impact Factor