Abstract
We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.
Highlights
More sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug
Identification of a GOGAT Small  Subunit Gene in E. histolytica upon L-Cysteine Supplementation—Upon analysis of the transcriptome of E. histolytica trophozoites cultured in medium supplemented with L-cysteine, a highly up-regulated gene (XM_648481) was previously identified
The genes were designated as E. histolytica NADPH-dependent oxidoreductase 1 and 2 [EhNO1 (XM_651905) and EhNO2 (XM_648481)] because the encoded proteins lacked glutamate synthase activity, they showed robust NADPH-dependent oxidoreductase activity
Summary
Chemicals and Reagents—L-Cysteine, L-cystine, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, cytochrome c, iodonitrotetrazolium (INT), and metronidazole were purchased from Sigma. The reactions were initiated by the addition of 2 g of rEhNO protein to a mixture comprising 50 mM Tris-HCl (pH 8.0), 0.5 mM metronidazole, and 0.2 mM NADPH. Reaction mixtures containing 0.2 mM NADPH, 100 mM Tris-HCl (pH 7.5), 0.005–1 mM ferric ammonium citrate, and 2 g of rEhNO1 and -2 were used for the assays. Activity was measured by monitoring cytochrome c reduction at 550 nm (⑀ ϭ 21.1 mMϪ1 cmϪ1) in a reaction mixture containing 0.1 mM NADPH, 0.01– 0.5 M ferredoxin, 10 M cytochrome c, and 50 mM Tris-HCl buffer (pH 7.5). Generation of E. histolytica Transformants Overexpressing EhNO—The protein coding regions of EhNO1 and EhNO2 were amplified by PCR from cDNA using sense and antisense oligonucleotides containing appropriate restriction sites at the 5Ј end. After further washing with TBST, specific proteins were visualized and measured with a chemiluminescence detection system (Millipore) using Scion Image software (Scion Corp., Frederick, MD) [39]
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