A new type of peroxidase ("thiol peroxidase"; TPx) having cysteine as the primary site of catalysis has been discovered from prokaryotes to eukaryotes. In addition to two yeast TPx isoforms (TSA I and TSA II/AHPC1) previously described, three additional TPx homologues were identified by analysis of the open reading frame data base for Saccharomyces cerevisiae. Three novel isoforms showed a distinct thiol peroxidase activity supported by thioredoxin, and appeared to be distinctively localized in cytoplasm, mitochondria, and nucleus.

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...

Background: Peroxiredoxins (Prxs) are a novel group of peroxidases containing high antioxidant efficiency. The mammalian Prx family has six distinct members (Prx I-VI) in various subcellular locations, including peroxisomes and mitochondria, places where oxidative stress is most evident. The function of Prx I in particular has been implicated in regulating cell proliferation, differentiation, and apoptosis. Since thioredoxin1 (Trx1) as an electron donor is functionally associated with Prx I, we investigated levels of expression of both Prx I and Trx1.

Yeast nucleus-localized thiol peroxidase (nTPx) was characterized as a functional peroxidase. There are two cysteine residues in nTPx. Replacement of Cys-106 or Cys-111 with serine resulted in a complete loss of thioredoxin-linked peroxidase activity. However, when their activities were measured in terms of the ability to inhibit oxidation of glutamine synthetase, C111S showed the same antioxidant activity as the wild type protein. SDS-PAGE gel analysis revealed that only C111S existed as the dimer form. In addition to the identification of Cys-106 as the primary catalytic site, these data suggest the formation of the intradisulfide bond as a part of the catalytic cycle between nTPx and thioredoxin. nTPx preferentially reduced alkyl-hydroperoxides rather than H 2 O 2 . Furthermore, a nTPx mutant strain showed higher sensitivity toward alkyl-hydroperoxide than hydrogen peroxide. Also, reduction of the viability of nTPx mutant strain against various oxidants supports an in vivo antioxidant role for nTPx. nTPx transcriptional activity was not significantly detectable in log phase yeast, but the activity was exponentially increased after the diauxic shift. The transcriptional activity was highly induced even in the log phase yeast grown in nonfermentable carbon source. Deletion of Tor1p, Ras1p, and Ras2p resulted in considerable induction when compared with their parent strains, demonstrating the activation of the transcription of nTPx gene at the diauxic shift. Transcription of nTPx gene was induced in response to oxidative stress. Viability of a stationary phase nTPx mutant was considerably reduced compared with the isogenic strain. Collectively, these data demonstrate that nTPx is a thiol peroxidase family acting as alkyl-hydroperoxide reductase in the nucleus during post-diauxic growth.