Alkyl Hydroperoxide

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Leslie B Poole - One of the best experts on this subject based on the ideXlab platform.

  • identification of cysteine sulfenic acid in ahpc of Alkyl Hydroperoxide reductase
    Methods in Enzymology, 2002
    Co-Authors: Leslie B Poole, Holly R Ellis
    Abstract:

    Summary C165S AhpC in its sulfenate (Cys-SO−) and presumed thiolate (Cys-S−) forms at pH 7 (pKa for sulfenic acid about pH 6.1) exhibit low extinction absorbance bands around 367 and 324 nm, respectively. Sulfenic acid content of the protein can be assessed by its reactivity with the chromophoric TNB anion. Using this technique, H2O2 titrations of C165S AhpC give a maximum of about 1 SOH per subunit on addition of 1.0 to 1.2 equivalents of H2O2. Cys46-SO− is moderately air stable at neutral pH and room temperature and is oxidized at a steady rate of about 10% per half hour. Cys46-SO− of C165S AhpC is reduced in the presence of catalytic amounts of AhpF by ∼1 equivalent of NADH to regenerate the Cys46-S− species. NBD chloride is extremely useful as a trapping agent for cysteine sulfenic acid. The Cys46-S(O)-NBD adduct absorbs maximally at 347 nm and is 16 amu larger than the Cys46-S-NBD adduct (γmax = 420 nm) as shown by ESI-MS. Other electrophilic thiol reagents also react with Cys46-SO−; however, iodoacetamide and N-ethylmaleimide reactivities are much lower with Cys46-SO− than with Cys46-S−. These methods are applicable to other sulfenic acid-containing proteins, although in some cases the proteins must be denatured in order to provide accessibility of this species toward labeling agents.

  • streptococcus mutans h2o2 forming nadh oxidase is an Alkyl Hydroperoxide reductase protein
    Free Radical Biology and Medicine, 2000
    Co-Authors: Leslie B Poole, Masako Higuchi, Mamoru Shimada, Marco Li Calzi, Yoshiyuki Kamio
    Abstract:

    Abstract Nox-1 from Streptococcus mutans , the bacteria which cause dental caries, was previously identified as an H 2 O 2 -forming reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nox-1 is homologous with the flavoprotein component, AhpF, of Salmonella typhimurium Alkyl Hydroperoxide reductase. A partial open reading frame upstream of nox 1, homologous with the other (peroxidase) component, ahpC , from the S. typhimurium system, was also identified. We report here the complete sequence of S. mutans ahpC . Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a cysteine-based peroxidase system in S. mutans , catalyzing the NADH-dependent reduction of organic Hydroperoxides or H 2 O 2 to their respective alcohols and/or H 2 O. These proteins also catalyze the four-electron reduction of O 2 to H 2 O, clarifying the role of Nox-1 as a protective protein against oxygen toxicity. Major differences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts of flavin semiquinone and a more prominent FADH 2 to NAD + charge transfer absorbance band stabilized by Nox-1; and (iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1. Although Nox-1 and AhpC from S. mutans were shown to play a protective role against oxidative stress in vitro and in vivo in Escherichia coli , the lack of a significant effect on deletion of these genes from S. mutans suggests the presence of additional antioxidant proteins in these bacteria.

  • requirement for the two ahpf cystine disulfide centers in catalysis of peroxide reduction by Alkyl Hydroperoxide reductase
    Biochemistry, 1997
    Co-Authors: Marco Li Calzi, Leslie B Poole
    Abstract:

    AhpF, the Alkyl Hydroperoxide reductase component which transfers electrons from pyridine nucleotides to the peroxidase protein, AhpC, possesses two redox-active disulfide centers in addition to one FAD per subunit; the primary goal of these studies has been to test for the requirement of one or both of these disulfide centers in catalysis. Two half-cystine residues of one center (Cys 345Cys348) align with those of the homologous Escherichia coli thioredoxin reductase (TrR) sequence (Cys135Cys138), while the other two (Cys129Cys132) reside in the additional N-terminal region of AhpF which has no counterpart in TrR. We have employed site-directed mutagenesis techniques to generate four mutants of AhpF, including one which removes the N-terminal disulfide (Ser129Ser132) and three which perturb the TrR-like disulfide center (Ser345Ser348, Ser345Cys348, and Cys345Ser348). Fluorescence, absorbance, and circular dichroism spectra show relatively small perturbations for mutations at the disulfide center proximal to the flavin (Cys345Cys348) and no changes for the Ser129Ser132 mutant; identical circular dichroism spectra in the ultraviolet region indicate unchanged secondary structures in all mutants studied. Oxidase and transhydrogenase activities are preserved in all mutants, indicating no role for cystine redox centers in these activities. Both DTNB and AhpC reduction by AhpF are dramatically affected by each of these mutations, dropping to less than 5% for DTNB reductase activity and to less than 2% for peroxidase activity in the presence of AhpC. Reductive titrations confirm the absence of one redox center in each mutant; even in the absence of Cys345Cys348, the N-terminal redox center can be reduced, although only slowly. These results emphasize the necessity for both redox-active disulfide centers in AhpF for catalysis of disulfide reductase activity and support a direct role for Cys 129Cys132 in mediating electron transfer between Cys345Cys348 and the AhpC active-site disulfide.

  • roles for the two cysteine residues of ahpc in catalysis of peroxide reduction by Alkyl Hydroperoxide reductase from salmonella typhimurium
    Biochemistry, 1997
    Co-Authors: Holly R Ellis, Leslie B Poole
    Abstract:

    The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the Alkyl Hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic Hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.

  • flavin dependent Alkyl Hydroperoxide reductase from salmonella typhimurium 2 cystine disulfides involved in catalysis of peroxide reduction
    Biochemistry, 1996
    Co-Authors: Leslie B Poole
    Abstract:

    The two-component Alkyl Hydroperoxide reductase enzyme system from Salmonella typhimurium catalyzes the pyridine nucleotide-dependent reduction of Alkyl Hydroperoxide and hydrogen peroxide substrates. This system is composed of a flavoenzyme, AhpF, which is related to the disulfide-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor. We have demonstrated that NADH-linked reduction of AhpF under anaerobic conditions converts two cystine disulfide centers to their dithiol forms. The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl Hydroperoxide. Disulfide bridges within oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys489, exist as free thiol groups in oxidized AhpF and play no role in catalysis. Removal of the N-terminal 202-amino acid segment containing the Cys129-Cys132 disulfide center obliterates the ability of AhpF to transfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC. NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin reduction; the reduction potentials of the disulfide centers are thus appropriately poised for electron transfer from NADH and flavin to disulfide-containing substrates (AhpC or DTNB), and ultimately to peroxides from AhpC. Blue, neutral flavin semiquinone is also generated in high yields during reductive titrations (91% yield during dithionite titrations), although the relatively slow formation of this species indicates its catalytic incompetence. A long wavelength absorbance band beyond 900 nm attributable to an FADH2-->NAD+ charge transfer interaction is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved in the catalytic cycle. A catalytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed.

Youichi Niimura - One of the best experts on this subject based on the ideXlab platform.

  • purified thioredoxin reductase from o2 sensitive bifidobacterium bifidum degrades h2o2 by interacting with Alkyl Hydroperoxide reductase
    Anaerobe, 2019
    Co-Authors: Takumi Satoh, Youichi Niimura, Mitsunori Todoroki, Kazuya Kobayashi, Shinji Kawasaki
    Abstract:

    Abstract Bifidobacterium is beneficial for host health and exhibits different O2 sensitivity levels among species or strains via unknown mechanisms. Bifidobacterium bifidum JCM1255T, a type species of Bifidobacterium, is an O2-sensitive bacterium that can grow under low-O2 (5%) conditions, and the growth of this species is inhibited under high-O2 conditions (10% ∼) with accumulation of H2O2. We previously reported that NADH or NAD(P)H oxidase-active fractions were detected during purification using microaerobically grown B. bifidum cells, and the active enzyme was purified from the NADH oxidase-active fraction. The purified enzyme was identified as b-type dihydroorotate dehydrogenase (DHODb) and characterized as a dominant H2O2 producer in B. bifidum. In this study, we performed further purification of the enzyme from the NAD(P)H oxidase-active fraction and characterized the purified enzyme as a part of the H2O2 degradation system in B. bifidum. This purified enzyme was identified as thioredoxin reductase (TrxR); the NAD(P)H oxidase activity of this enzyme was not expressed in anaerobically grown B. bifidum, and mRNA expression was induced by O2 exposure. Furthermore, the purified B. bifidum TrxR interacted with recombinant Alkyl Hydroperoxide reductase (rAhpC) and exhibited NAD(P)H peroxidase activity. These results suggest that TrxR responds to O2 and protects B. bifidum from oxidative stress by degrading H2O2 via the TrxR-AhpC system.

  • Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria
    Journal of Bacteriology, 2001
    Co-Authors: Yoshitaka Nishiyama, Shinji Kawasaki, Vincent Massey, Kouji Takeda, Junichi Sato, Toshihiro Watanabe, Youichi Niimura
    Abstract:

    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 Vmax 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.

  • reaction mechanism of amphibacillus xylanus nadh oxidase Alkyl Hydroperoxide reductase flavoprotein
    Journal of Biological Chemistry, 1996
    Co-Authors: Youichi Niimura, Vincent Massey
    Abstract:

    Abstract NADH oxidase from Amphibacillus xylanus is a potent Alkyl Hydroperoxide reductase in the presence of the small disulfide-containing protein (AhpC) of Salmonella typhimurium. In the presence of saturating AhpC, kcat values for reduction of Hydroperoxides are approximately 180 s−1, and the double mutant flavoprotein enzyme C337S/C340S cannot support Hydroperoxide reduction (Niimura, Y., Poole, L. B., and Massey, V. (1995) J. Biol. Chem. 270, 25645-25650). Kinetics of reduction of wild-type and mutant enzymes are reported here with wild-type enzyme; reduction by NADH was triphasic, with consumption of 2.6 equivalents of NADH, consistent with the known composition of one FAD and two disulfides per subunit. Rate constants for the first two phases (each approximately 200 s−1) where FAD and one disulfide are reduced are slightly greater than kcat values for AhpC-linked Hydroperoxide reduction. The rate constant for the third phase (reduction to the 6-electron level) is too small for catalysis. Only the first phase of the wild-type enzyme occurs with the mutant enzyme. These results and the stoichiometry of NADH consumption indicate Cys337 and Cys340 as the active site disulfide of the flavoprotein and that electrons from FADH2 must pass through this disulfide to reduce the disulfide of AhpC.

  • amphibacillus xylanus nadh oxidase and salmonella typhimurium Alkyl Hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both Alkyl Hydroperoxide and hydrogen peroxide in the presence of s typhimurium Alkyl hydr
    Journal of Biological Chemistry, 1995
    Co-Authors: Youichi Niimura, Leslie B Poole, Vincent Massey
    Abstract:

    The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide. The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the Alkyl-Hydroperoxide reductase from Salmonella typhimurium. AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein. In the presence of the 22-kDa colorless component (AhpC) of the Salmonella Alkyl-Hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water. Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene Hydroperoxide. Both enzymes' Km values for hydrogen peroxide, cumene Hydroperoxide, and NADH are so low that they could not be determined accurately. Vmax values for hydrogen peroxide or cumene Hydroperoxide reduction are > 10,000 min(-1) at 25 degrees C. These values are almost the same as the reduction rate of the flavoprotein component by NADH. The involvement in catalysis of a redox-active disulfide of the A. xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C40SC337S/C340S. Very little activity for hydrogen peroxide or cumene Hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S). Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional Alkyl-Hydroperoxide reductase system within these catalase-lacking bacteria.

Skorn Mongkolsuk - One of the best experts on this subject based on the ideXlab platform.

  • a suppressor of the menadione hypersensitive phenotype of a xanthomonas campestris pv phaseoli oxyr mutant reveals a novel mechanism of toxicity and the protective role of Alkyl Hydroperoxide reductase
    Journal of Bacteriology, 2003
    Co-Authors: Paiboon Vattanaviboon, Wirongrong Whangsuk, Skorn Mongkolsuk
    Abstract:

    We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyRXp). The oxyRR2Xp mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H2O2 and tert-butyl Hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2Xp mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic Hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2Xp mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyRXp mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that Alkyl Hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris.

  • evaluation of the roles that Alkyl Hydroperoxide reductase and ohr play in organic peroxide induced gene expression and protection against organic peroxides in xanthomonas campestris
    Biochemical and Biophysical Research Communications, 2002
    Co-Authors: Paiboon Vattanaviboon, Warunya Panmanee, Chananat Klomsiri, Saovanee Dharmsthiti, Wirongrong Whangsuk, Skorn Mongkolsuk
    Abstract:

    Alkyl Hydroperoxide reductase (ahpC) and organic Hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic Hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl Hydroperoxide (tBOOH) while the former was more efficient at degrading cumene Hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.

  • a xanthomonas Alkyl Hydroperoxide reductase subunit c ahpc mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes
    Journal of Bacteriology, 2000
    Co-Authors: Paiboon Vattanaviboon, Wirongrong Whangsuk, Skorn Mongkolsuk, Suvit Loprasert, Mayuree Fuangthong
    Abstract:

    Alkyl Hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for Alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H2O2 killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.

Vincent Massey - One of the best experts on this subject based on the ideXlab platform.

  • Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria
    Journal of Bacteriology, 2001
    Co-Authors: Yoshitaka Nishiyama, Shinji Kawasaki, Vincent Massey, Kouji Takeda, Junichi Sato, Toshihiro Watanabe, Youichi Niimura
    Abstract:

    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 Vmax 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.

  • reaction mechanism of amphibacillus xylanus nadh oxidase Alkyl Hydroperoxide reductase flavoprotein
    Journal of Biological Chemistry, 1996
    Co-Authors: Youichi Niimura, Vincent Massey
    Abstract:

    Abstract NADH oxidase from Amphibacillus xylanus is a potent Alkyl Hydroperoxide reductase in the presence of the small disulfide-containing protein (AhpC) of Salmonella typhimurium. In the presence of saturating AhpC, kcat values for reduction of Hydroperoxides are approximately 180 s−1, and the double mutant flavoprotein enzyme C337S/C340S cannot support Hydroperoxide reduction (Niimura, Y., Poole, L. B., and Massey, V. (1995) J. Biol. Chem. 270, 25645-25650). Kinetics of reduction of wild-type and mutant enzymes are reported here with wild-type enzyme; reduction by NADH was triphasic, with consumption of 2.6 equivalents of NADH, consistent with the known composition of one FAD and two disulfides per subunit. Rate constants for the first two phases (each approximately 200 s−1) where FAD and one disulfide are reduced are slightly greater than kcat values for AhpC-linked Hydroperoxide reduction. The rate constant for the third phase (reduction to the 6-electron level) is too small for catalysis. Only the first phase of the wild-type enzyme occurs with the mutant enzyme. These results and the stoichiometry of NADH consumption indicate Cys337 and Cys340 as the active site disulfide of the flavoprotein and that electrons from FADH2 must pass through this disulfide to reduce the disulfide of AhpC.

  • amphibacillus xylanus nadh oxidase and salmonella typhimurium Alkyl Hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both Alkyl Hydroperoxide and hydrogen peroxide in the presence of s typhimurium Alkyl hydr
    Journal of Biological Chemistry, 1995
    Co-Authors: Youichi Niimura, Leslie B Poole, Vincent Massey
    Abstract:

    The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide. The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the Alkyl-Hydroperoxide reductase from Salmonella typhimurium. AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein. In the presence of the 22-kDa colorless component (AhpC) of the Salmonella Alkyl-Hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water. Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene Hydroperoxide. Both enzymes' Km values for hydrogen peroxide, cumene Hydroperoxide, and NADH are so low that they could not be determined accurately. Vmax values for hydrogen peroxide or cumene Hydroperoxide reduction are > 10,000 min(-1) at 25 degrees C. These values are almost the same as the reduction rate of the flavoprotein component by NADH. The involvement in catalysis of a redox-active disulfide of the A. xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C40SC337S/C340S. Very little activity for hydrogen peroxide or cumene Hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S). Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional Alkyl-Hydroperoxide reductase system within these catalase-lacking bacteria.

Hinchung Wong - One of the best experts on this subject based on the ideXlab platform.

  • activities of Alkyl Hydroperoxide reductase subunits c1 and c2 of vibrio parahaemolyticus against different peroxides
    Applied and Environmental Microbiology, 2014
    Co-Authors: Chunhui Chung, Shinyuan Fen, Hinchung Wong
    Abstract:

    Alkyl Hydroperoxide reductase subunit C gene (ahpC) functions were characterized in Vibrio parahaemolyticus, a commonly occurring marine food-borne enteropathogenic bacterium. Two ahpC genes, ahpC1 (VPA1683) and ahpC2 (VP0580), encoded putative two-cysteine peroxiredoxins, which are highly similar to the homologous proteins of Vibrio vulnificus. The responses of deletion mutants of ahpC genes to various peroxides were compared with and without gene complementation and at different incubation temperatures. The growth of the ahpC1 mutant and ahpC1 ahpC2 double mutant in liquid medium was significantly inhibited by organic peroxides, cumene Hydroperoxide and tert-butyl Hydroperoxide. However, inhibition was higher at 12°C and 22°C than at 37°C. Inhibiting effects were prevented by the complementary ahpC1 gene. Inconsistent detoxification of H2O2 by ahpC genes was demonstrated in an agar medium but not in a liquid medium. Complementation with an ahpC2 gene partially restored the peroxidase effect in the double ahpC1 ahpC2 mutant at 22°C. This investigation reveals that ahpC1 is the chief peroxidase gene that acts against organic peroxides in V. parahaemolyticus and that the function of the ahpC genes is influenced by incubation temperature.

  • roles of Alkyl Hydroperoxide reductase subunit c ahpc in viable but nonculturable vibrio parahaemolyticus
    Applied and Environmental Microbiology, 2013
    Co-Authors: Henwei Wang, Chunhui Chung, Hinchung Wong
    Abstract:

    Alkyl Hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for the detoxification of reactive oxygen species that form in bacterial cells or are derived from the host; thus, AhpC facilitates the survival of pathogenic bacteria under environmental stresses or during infection. This study investigates the role of AhpC in the induction and maintenance of a viable but nonculturable (VBNC) state in Vibrio parahaemolyticus. In this investigation, ahpC1 (VPA1683) and ahpC2 (VP0580) were identified in chromosomes II and I of this pathogen, respectively. Mutants with deletions of these two ahpC genes and their complementary strains were constructed from the parent strain KX-V231. The growth of these strains was monitored on tryptic soy agar–3% NaCl in the presence of the extrinsic peroxides H2O2 and tert-butyl Hydroperoxide (t-BOOH) at different incubation temperatures. The results revealed that both ahpC genes were protective against t-BOOH, while ahpC1 was protective against H2O2. The protective function of ahpC2 at 4°C was higher than that of ahpC1. The times required to induce the VBNC state (4.7 weeks) at 4°C in a modified Morita mineral salt solution with 0.5% NaCl and then to maintain the VBNC state (4.7 weeks) in an ahpC2 mutant and an ahpC1 ahpC2 double mutant were significantly shorter than those for the parent strain (for induction, 6.2 weeks; for maintenance, 7.8 weeks) and the ahpC1 mutant (for induction, 6.0 weeks; for maintenance, 8.0 weeks) (P < 0.03). Complementation with an ahpC2 gene reversed the effects of the ahpC2 mutation in shortening the times for induction and maintenance of the VBNC state. This investigation identified the different functions of the two ahpC genes and confirmed the particular role of ahpC2 in the VBNC state of V. parahaemolyticus.