scholarly journals Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica

1997 ◽  
Vol 326 (3) ◽  
pp. 785-789 ◽  
Author(s):  
Iris BRUCHHAUS ◽  
Symi RICHTER ◽  
Egbert TANNICH
Keyword(s):  
Hydrogen Peroxide ◽  
Entamoeba Histolytica ◽  
Recombinant Proteins ◽  
Peroxidase Activity ◽  
Cumene Hydroperoxide ◽  
High Sensitivity ◽  
Coli Strain ◽  
Amino Acid Residues ◽  
Genes Encoding ◽  
Thiol Peroxidase

The 29 kDa protein of Entamoeba histolytica (Eh29), as well as a truncated variant of this protein, which lacks a cysteine-rich N-terminal region of 40 amino acid residues (Eh29mut), were recombinantly expressed in Escherichia coli and purified to homogeneity. Both recombinant proteins (recEh29, recEh29mut) were found to have hydrogen peroxide (H2O2)-removing activity, but recEh29 was twice as active as recEh29mut. For the consumption of exogenous H2O2, activity was dependent on the presence of reducing equivalents, such as dithiothreitol (DTT), indicating that Eh29 constitutes a thiol-dependent peroxidase. DTT was not required to remove H2O2 by recEh29 or recEh29mut when H2O2 was generated enzymically by the E. histolytica NADPH:flavin oxidoreductase. This enzyme produces H2O2 under aerobic conditions and simultaneously serves as a hydrogen donor for Eh29. Peroxidase activity of the recombinant proteins was further supported by complementation of an E. coli strain that lacks the entire alkyl hydroperoxide reductase locus. The high sensitivity of these bacteria against cumene hydroperoxide was significantly reduced by the introduction of the genes encoding recEh29 or recEh29mut. Using antisera raised against the recombinant proteins, native Eh29 was localized within the cytoplasm of the amoebae. In addition, the antisera reacted with proteins of E. histolytica lysates with apparent molecular masses of 35 kDa and 160–300 kDa. All of them exhibited thiol-peroxidase activity.

A hydrogen peroxide electrode assay to measure thiol peroxidase activity for organoselenium and organotellurium drugs

2012 ◽  
Vol 429 (2) ◽  
pp. 103-107 ◽  
Author(s):  
Niroshini M. Giles ◽  
Sweta Kumari ◽  
Rosemary A. Stamm ◽  
Siddharth Patel ◽  
Gregory I. Giles
Keyword(s):  
Hydrogen Peroxide ◽  
Peroxidase Activity ◽  
Thiol Peroxidase

scholarly journals A Hydrogen Peroxide-Forming NADH Oxidase That Functions as an Alkyl Hydroperoxide Reductase in Amphibacillus xylanus

2000 ◽  
Vol 182 (18) ◽  
pp. 5046-5051 ◽  
Author(s):  
Youichi Niimura ◽  
Yoshitaka Nishiyama ◽  
Daisuke Saito ◽  
Hirokazu Tsuji ◽  
Makoto Hidaka ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Peroxidase Activity ◽  
Disulfide Bonds ◽  
Nadh Oxidase ◽  
Reductase Activity ◽  
Accurate Determination ◽  
Cumene Hydroperoxide ◽  
Order Rate Constant ◽  
Alkyl Hydroperoxide ◽  
Growing Conditions

ABSTRACT The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with β-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containingSalmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812–5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 × 106 M−1 s−1. Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH2 reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH2 is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V max values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s−1. The Km values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.

scholarly journals Homocysteine peroxidase activity in rat blood plasma: stoichiometry and enzymatic character of the reaction

2013 ◽  
Vol 59 (6) ◽  
pp. 636-643 ◽  
Author(s):  
A.V. Razygraev
Keyword(s):  
Hydrogen Peroxide ◽  
Glutathione Peroxidase ◽  
Blood Plasma ◽  
Peroxidase Activity ◽  
Protein Fraction ◽  
Specific Activity ◽  
Rat Blood ◽  
Significant Acceleration ◽  
Thiol Peroxidase ◽  
Reaction Stoichiometry

Recently it was shown that the presence of rat blood plasma (as well as of erythrocyte hemolysate) in the reaction mixture containing 43 mM Tris-HCl-buffer (pH 8.5), 0.29 mM EDTA, 19.2 mM sodium azide, 1 mM DL-homocysteine (Hcy), and 198 mM hydrogen peroxide (incubation at 37°C) results in a significant acceleration of the decrease in Hcy concentration caused by addition of H O . In this paper, we present data indicating that the observed activity is the homocysteine:H O -oxidoreductase (homocysteine peroxidase) activity. It has been found that the level of H O -dependent Hcy decrease observed in the presence of blood plasma corresponds to homocysteine:H O -oxidoreductase reaction stoichiometry of 2:1 (mole ratio). The activity observed belongs to the protein fraction isolated by saturation with ammonium sulfate to 50%; the specific activity in this protein fraction is significantly higher than that in the whole plasma. The results confirm the hypothesis that the reaction between Hcy and H O at the presence of plasma is catalyzed by the protein component of plasma and this is the homocysteine peroxidase reaction. This activity is not associated with serum albumin, which is known to function as thiol peroxidase, and probably belongs to extracellular glutathione peroxidase (Gpx3).

An ESR study of the peroxidase reaction catalyzed by human methaemoglobin and methaemoglobin-haptoglobin complex

1991 ◽  
Vol 56 (4) ◽  
pp. 923-932
Author(s):  
Jana Stejskalová ◽  
Pavel Stopka ◽  
Zdeněk Pavlíček
Keyword(s):  
Hydrogen Peroxide ◽  
Ascorbic Acid ◽  
Amino Acid ◽  
Peroxidase Activity ◽  
Amino Acid Analysis ◽  
Superoxide Radical ◽  
Esr Spectra ◽  
The Other ◽  
Delocalized Electron

The ESR spectra of peroxidase systems of methaemoglobin-ascorbic acid-hydrogen peroxide and methaemoglobin-haptoglobin complex-ascorbic acid-hydrogen peroxide have been measured in the acetate buffer of pH 4.5. For the system with methaemoglobin an asymmetrical signal with g ~ 2 has been observed which is interpreted as the perpendicular region of anisotropic spectrum of superoxide radical. On the other hand, for the system with methaemoglobin-haptoglobin complex the observed signal with g ~ 2 is symmetrical and is interpreted as a signal of delocalized electron. After realization of three repeatedly induced peroxidase processes the ESR signal of the perpendicular part of anisotropic spectrum of superoxide radical is distinctly diminished, whereas the signal of delocalized electron remains practically unchanged. An amino acid analysis of methaemoglobin along with results of the ESR measurements make it possible to derive a hypothesis about the role of haptoglobin in increasing of the peroxidase activity of methaemoglobin.

scholarly journals RpoS-Dependent Stress Response and Exoenzyme Production in Vibrio vulnificus

2003 ◽  
Vol 69 (10) ◽  
pp. 6114-6120 ◽  
Author(s):  
A. Hülsmann ◽  
T. M. Rosche ◽  
I.-S. Kong ◽  
H. M. Hassan ◽  
D. M. Beam ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Stress Response ◽  
Mutant Strain ◽  
Environmental Changes ◽  
Vibrio Vulnificus ◽  
High Sensitivity ◽  
Striking Difference ◽  
Wild Type ◽  
Hydrolytic Activities ◽  
In The Wild

ABSTRACT Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence.

Direct cloning of genes encoding novel xylanases from the human gut

2005 ◽  
Vol 51 (3) ◽  
pp. 251-259 ◽  
Author(s):  
Hidenori Hayashi ◽  
Takashi Abe ◽  
Mitsuo Sakamoto ◽  
Hiroki Ohara ◽  
Toshimichi Ikemura ◽  
...  
Keyword(s):  
Intestinal Bacteria ◽  
Fecal Microbiota ◽  
Coli Strain ◽  
Amino Acid Sequences ◽  
Self Organizing Map ◽  
Human Gut ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Ph Range ◽  
Self Organizing

The aim of this study was to identify a novel 1,4-β-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43 552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombinant Escherichia coli strain and characterized. This enzyme was optimally active at 40 °C and stable up to 50 °C at pH 6.5 and over the pH range 4.0–11.0 at 25 °C. In addition, 2 ORFs (ORF1 and ORF2) were identified upstream of xyn10A. These results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.Key words: xylanase, human gut, fecal microbiota, phylogenetic analysis, self-organizing map.

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