Thioredoxin-linked "Thiol Peroxidase" from Periplasmic Space of Escherichia coli

Abstract

Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli. These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species. Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol. Analysis of the amino-terminal amino acid sequences and the immunoblots between 24-and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E. coli alkyl hydroperoxide reductase (AhpC/ AhpF). We isolated and sequenced an E. coli genomic DNA fragment that encodes 20-kDa protein. Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology.A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme. The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth. The 20-kDa protein resides mainly in the periplasmic space of E. coli, whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This 20-kDa protein is thus named "thiol peroxidase," which could act as an antioxidant enzyme removing peroxides or H 2 O 2 within the catalase-and peroxidase-deficient periplasmic space of E. coli.

Vibrio cholerae Thiol Peroxidase-Glutaredoxin Fusion Is a 2-Cys TSA/AhpC Subfamily Acting as a Lipid Hydroperoxide Reductase

“…The supernatants clarified by centrifugation were used for purification of proteins. The recombinant proteins were purified according to the methods previously reported (8,9,17,20).…”

“…For AhpC, reduction of peroxide is achieved by a specialized electron donor, AhpF (12), whereas BCP and p20 receive electrons from a reducing system composed of Trx and Trx reductase (11,13). P20 has been characterized as a periplasmic protein (9) that has been reported to exist in Gram-negative bacteria such as Escherichia coli (9), whereas AhpC and BCP as cytoplasmic proteins have been found in all species of bacteria (14).…”

“…To alleviate the oxidative damage of these compounds, bacterium induces the synthesis of a variety of antioxidant defense enzymes, such as hydroperoxidases (catalases) I and II (gene products of katG and katE, respectively), that decompose H 2 O 2 (5) and superoxide dismutases (manganese superoxide dismutase, sodA; iron superoxide dismutase, sodB; copper-zinc superoxide dismutase, sodC) that eliminate superoxide anion (6). Additional defenses in bacteria against alkyl and lipid hydroperoxides are suggested to be provided by alkyl hydroperoxide reductase (AhpC) (7), bacterioferritin-comigratory protein (BCP) (8), and periplasmic thiol peroxidase (p20) (9). AhpC and BCP are all bacterial members of the ubiquitous thiol peroxidase (TPx) (TSA/AhpC) family (7)(8)(9)(10)(11).…”

“…Research over the past decades has led to the characterization of a new family of peroxidases, collectively called peroxyredoxins, TSA/AhpC family (20) or TPx family (8,9,21,34). TPx proteins can be divided into two subgroups, 2-Cys TPx (22) and 1-Cys TPx (23,35), depending on the number of conserved cysteines.…”

“…Additional defenses in bacteria against alkyl and lipid hydroperoxides are suggested to be provided by alkyl hydroperoxide reductase (AhpC) (7), bacterioferritin-comigratory protein (BCP) (8), and periplasmic thiol peroxidase (p20) (9). AhpC and BCP are all bacterial members of the ubiquitous thiol peroxidase (TPx) (TSA/AhpC) family (7)(8)(9)(10)(11). For AhpC, reduction of peroxide is achieved by a specialized electron donor, AhpF (12), whereas BCP and p20 receive electrons from a reducing system composed of Trx and Trx reductase (11,13).…”