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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.

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, Vincent Massey, Kouji Takeda, Shinji Kawasaki, 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.

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, Skorn Mongkolsuk, Wirongrong Whangsuk

    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, Skorn Mongkolsuk, Wirongrong Whangsuk, Warunya Panmanee, Chananat Klomsiri, Saovanee Dharmsthiti

    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, Skorn Mongkolsuk, Wirongrong Whangsuk, 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.