Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica.
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.
Bruchhaus
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I
Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.
Richter
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S
Tannich
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E
D000818:Animals; D004748:Entamoeba histolytica / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide