Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica.
The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN.
Bruchhaus
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Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.
Richter
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Tannich
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D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology*; D004926:Escherichia coli; D038181:FMN Reductase; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D004220:Disulfides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C095052:disulfide oxidoreductase, Entamoeba; D010100:Oxygen