Thioredoxin-dependent Hydroperoxide Peroxidase Activity of Bacterioferritin Comigratory Protein (BCP) as a New Member of the Thiol-specific Antioxidant Protein (TSA)/Alkyl Hydroperoxide Peroxidase C (AhpC) Family

Abstract

Escherichia coli bacterioferritin comigratory protein (BCP), a putative bacterial member of the TSA/AhpC family, was characterized as a thiol peroxidase. BCP showed a thioredoxin-dependent thiol peroxidase activity. BCP preferentially reduced linoleic acid hydroperoxide rather than H 2 O 2 and t-butyl hydroperoxide with the use of thioredoxin as an in vivo immediate electron donor. The value of V max /K m of BCP for linoleic acid hydroperoxide was calculated to be 5-fold higher than that for H 2 O 2 , implying that BCP has a selective capability to reduce linoleic acid hydroperoxide. Replacement of Cys-45 with serine resulted in the complete loss of thiol peroxidase activity, suggesting that BCP is a new bacterial member of TSA/AhpC family having a conserved cysteine as the primary site of catalysis. BCP exists as a monomer, and its functional Cys-45 appeared to exist as cysteine sulfenic acid. The expression level of BCP gradually elevated during exponential growth until mid-log phase growth, beyond which the expression level was decreased. BCP was induced 3-fold by the oxidative stress given by changing the growth conditions from the anaerobic to aerobic culture. Bcp null mutant grew more slowly than its wild type in aerobic culture and showed the hypersensitivity toward various oxidants such as H 2 O 2 , t-butyl hydroperoxide, and linoleic acid hydroperoxide. The peroxide hypersensitivity of the null mutant could be complemented by the expression of bcp gene. Taken together, these data suggest that BCP is a new member of thioredoxin-dependent TSA/ AhpC family, acting as a general hydroperoxide peroxidase. Reactive oxygen species (ROS)1 are potent oxidants capable of damaging all cellular constituents. To protect against the toxicity of ROS, aerobic organisms are equipped with an array of defense mechanism (1). In mammals, glutathione peroxidase acts a general hydroperoxide to reduce H 2 O 2 and alkyl hydroperoxides including fatty acid hydroperoxide (1, 2). The alkyl hydroperoxide reductase (AhpC)/TSA family is a new type of peroxidase that has a conserved cysteine as the primary site of catalysis instead of the selenocysteine of glutathione peroxidase. The new type of peroxidase that has been discovered from prokaryotes to eukaryotes (3, 4) reduces hydroperoxides with the use of thioredoxin and other thiol-containing reducing agents (5-7).Only two bacterial member of the new peroxidase family have been reported, although at least five types of thiol peroxidase exist in eukaryotic cell. In Escherichia coli, AhpC was reported to preferentially reduce alkyl hydroperoxide with electrons provided by either NADH or NADPH via the AhpF52 (8). Recently, we reported a novel type of peroxidase, p20, that acts as an antioxidant to remove peroxides such as H 2 O 2 and alkyl hydroperoxide in the periplasmic space of E. coli (9).The similarity of primary structure between bacterioferritin comigratory protein (BCP) and the TSA/AhpC family suggested that BCP could be another new member of the family. However, the function of...

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).…”

“…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).…”

Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein fromThermotoga maritima

“…Nevertheless, an intramolecular disulfide bridge can be formed between these two cysteines in plant enzymes and, as the presence of these two cysteines leads to a more active enzyme, the physiological regeneration mechanism used by the enzyme would preferably involve the formation of this particular disulfide bridge. In E. coli, it has been shown that a mutated protein possessing only the peroxidatic cysteine is still active and is regenerated by thioredoxin (Jeong et al, 2000). Again, the classification is not adapted to this class of Prx as no intramolecular disulfide bridge can be formed in proteins that possess only one conserved cysteine.…”