Cloning, Overexpression, and Characterization of Peroxiredoxin and NADH Peroxiredoxin Reductase from Thermus aquaticus

Logan, Catriona; Mayhew, Stephen G.

doi:10.1074/jbc.m004161200

Cited by 20 publications

(19 citation statements)

References 42 publications

(46 reference statements)

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“…No other peroxide-scavenging enzyme, catalase or peroxidase, so far studied has been reported to show such high turnover numbers and low K m values for both hydrogen peroxide and alkyl hydroperoxide as described here (4,6,8,11,17,32,35,47). A recent report also describes the isolation and function of peroxiredoxin and peroxiredoxin reductase from a thermophile, Thermus aquaticus (18). The flavoprotein component, PrxR, was originally isolated as an NADH oxidase (7).…”

Section: Discussionmentioning

confidence: 72%

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Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria

et al. 2001

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Amphibacillus xylanus and Sporolactobacillus inulinus NADH oxidases belonging to the peroxiredoxin oxidoreductase family show extremely high peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of the small disulfide redox protein, AhpC (peroxiredoxin). In order to investigate the distribution of this enzyme system in bacteria, 15 bacterial strains were selected from typical aerobic, facultatively anaerobic, and anaerobic bacteria. AhpC-linked alkyl hydroperoxide reductase activities were detected in most of the tested strains, and especially high activities were shown in six bacterial species that grow well under aerobic conditions, including aerobic bacteria (Alcaligenes faecalis and Bacillus licheniformis) and facultatively anaerobic bacteria (Amphibacillus xylanus, Sporolactobacillus inulinus, Escherichia coli, and Salmonella enterica serovar Typhimurium). In the absence of AhpC, the purified enzymes from A. xylanus and S. inulinus catalyze the NADH-linked reduction of oxygen to hydrogen peroxide. Similar activities were observed in the cell extracts from each of these six strains. The cell extract of B. licheniformis revealed the highest AhpC-linked alkyl hydroperoxide reductase activity in the four strains, with V max values for hydrogen peroxide and alkyl hydroperoxides being similar to those for the enzymes from A. xylanus and S. inulinus. Southern blot analysis of the three strains probed with the A. xylanus peroxiredoxin reductase gene revealed single strong bands, which are presumably derived from the individual peroxiredoxin reductase genes. Single bands were also revealed in other strains which show high AhpC-linked reductase activities, suggesting that the NADH oxidases belonging to the peroxiredoxin oxidoreductase family are widely distributed and possibly play an important role both in the peroxide-scavenging systems and in an effective regeneration system for NAD in aerobically growing bacteria.NADH oxidases are found in several microorganisms (9,12,20,31,41) and have been purified from at least nine bacterial species (7,10,13,25,30,34,40,42,44). There are two types of NADH oxidase, H 2 O forming and hydrogen peroxide forming. We previously purified the hydrogen peroxide-forming NADH oxidases from aerobically grown Amphibacillus xylanus and Sporolactobacillus inulinus, both of which are facultatively anaerobic bacteria that lack a respiratory chain (25, 30). The physiological function of these enzymes was first thought to be the in vivo regeneration of NAD in aerobic metabolism of the bacteria (22-24, 30). The enzymes catalyze the reduction of oxygen by NADH to form hydrogen peroxide. However, in the presence of a 21-kDa disulfide-containing redox protein (AhpC), now commonly referred to as peroxiredoxin (Prx), the NADH oxidases also showed extremely high reductase activity for both hydrogen peroxide and alkyl hydroperoxides (26,(28)(29)(30). These NADH oxidases thus belong to a growing new family of peroxiredoxin oxidoreductases (PrxR) (38) and are involved...

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Section: Discussionmentioning

confidence: 72%

“…The flavoprotein component, PrxR, was originally isolated as an NADH oxidase (7). This system, however, shows much lower catalytic activity than the ones described above (18).…”

Section: Discussionmentioning

confidence: 95%

Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria

et al. 2001

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“…The peroxiredoxin assay was described previously (11,20). Spores or vegetative cells (1 ϫ 10 8 ) were pelleted by centrifugation and suspended in a reaction buffer that included H 2 O 2 .…”

Section: Methodsmentioning

confidence: 99%

Surface Layers of Clostridium difficile Endospores

et al. 2011

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Clostridium difficile is an important human pathogen and one where the primary cause of disease is due to the transmission of spores. We have investigated the proteins found in the outer coat layers of C. difficile spores of pathogenic strain 630 (CD630). Five coat proteins, CotA, CotB, CotCB, CotD, and CotE, were shown to be expressed on the outer coat layers of the spore. We demonstrate that purified spores carry catalase, peroxiredoxin, and chitinase activity and that this activity correlates with the predicted functions of three spore coat proteins identified here, CotCB, CotD, and CotE. CotCB and CotD are putative manganese catalases, and CotE is a novel bifunctional protein with peroxiredoxin activity at its amino terminus and chitinase activity at its carboxy terminus. These enzymes could play an important role in coat assembly by polymerizing protein monomers in the coat. CotE, in addition to a role in macromolecular degradation, could play an important role in inflammation, and this may be of direct relevance to the development of the gastrointestinal symptoms that accompany C. difficile infection. Although specific enzyme activity has not yet been assigned to the proteins identified here, this work provides the first detailed study of the C. difficile spore coat.

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“…The peroxiredoxin assay was carried out as described previously (32,33). Two hundred fifty micromolar ␤-NAD (NADH, N8129; Sigma), 500 M H 2 O 2 , 4 M thioredoxin (from E. coli, T0910; Sigma), and 0.1 M thioredoxin reductase (from E. coli, T7915; Sigma) were mixed with 1 ϫ 10 8 spores in 500 l of buffer [containing 0.3 mM EDTA, 0.5 M KH 2 PO 4 , and 150 mM (NH 4 ) 2 SO 4 ].…”

Section: Strainsmentioning

confidence: 99%

Functional Characterization of Clostridium difficile Spore Coat Proteins

et al. 2013

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dSpores of Clostridium difficile play a key role in the dissemination of this important human pathogen, and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly, with a clear misassembly of the outermost layers of the spore coat. The CotA protein is most probably subject to posttranslational modification and could play a key role in stabilizing the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore, although for the cotD, cotE, and sodA mutants, enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively that they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed to be enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. These were a manganese catalase, encoded by cotD, a superoxide dismutase (SOD), encoded by sodA, and a bifunctional enzyme with peroxiredoxin and chitinase activity, encoded by cotE. These enzymes being exposed on the spore surface would play a role in coat polymerization and detoxification of H 2 O 2 . Two additional proteins, CotF (a tyrosine-rich protein and potential substrate for SodA) and CotG (a putative manganese catalase) were shown to be located at the spore surface.

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Cloning, Overexpression, and Characterization of Peroxiredoxin and NADH Peroxiredoxin Reductase from Thermus aquaticus

Cited by 20 publications

References 42 publications

Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria

Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria

Surface Layers of Clostridium difficile Endospores

Functional Characterization of Clostridium difficile Spore Coat Proteins

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