Two atypical L-cysteine-regulated NADPH-dependent oxidoreductases involved in redox maintenance, L-cystine and iron reduction, and metronidazole activation in the enteric protozoan Entamoeba histolytica

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.

FIGURE 1.

Regulation of gene expression of EhNO isotypes in E. histolytica by extracellular l-cysteine concentration. E. histolytica trophozoites were cultured in normal (8 mm), l-cysteine-supplemented (18 mm), or deprived medium. The expression levels of the EhNO transcripts under l-cysteine-supplemented or -deprived conditions were normalized against those of RNA polymerase II and are shown as the -fold change expression of mRNA relative to that of trophozoites from the control (normal) culture. Error bars represent the S.E. of three independent experiments.

FIGURE 2.

Effects of extracellular l-cysteine concentrations on the amount of EhNO isotypes and intracellular l-cystine/l-cysteine concentrations. A, an immunoblot analysis of EhNO1 and -2 is shown. After trophozoites were cultured under normal or l-cysteine-supplemented conditions for 48 h, ∼15 μg of total cell lysate was electrophoresed on a 12% SDS-PAGE gel and subjected to an immunoblot assay with antibodies raised against EhNO1, EhNO2, or EhMPR1 as a control. The densitometric quantification of the reacted bands, shown in the right graph, was performed by Scion Image software, and the level of EhNO1, EhNO2, and EhMPR1 proteins was expressed in arbitrary units. Error bars represent the S.E. of three independent experiments. B, shown is intracellular l-cystine/cysteine concentrations under normal, l-cysteine-supplemented, and deprived conditions. l-Cystine/cysteine concentrations of the trophozoites cultivated for 48 h under the indicated conditions were analyzed by CE-MS. Error bars represent the S.E. of three independent experiments.

FIGURE 3.

Absorption spectra of rEhNO1 and rEhNO2 proteins. UV-visible absorption spectra of rEhNO1 (400 μg of protein) and rEhNO2 (200 μg) under non-reducing (solid lines) and sodium dithionite-reducing conditions (broken lines) are shown. The samples were reduced with a 10-fold molar excess of sodium dithionite.

FIGURE 4.

In vitro interactions of EhNO with ferredoxin. A, SDS-PAGE analysis of the complex of EhNO and ferredoxin is shown. Protein mixtures were incubated for 30 min with (even lane numbers) and without (odd lane numbers) 5 mm carbodiimide, electrophoresed on a 15% SDS-PAGE gel under reducing conditions, and then stained with Coomassie Brilliant Blue R250. The examined protein mixtures were as follows: lanes 1 and 2, spinach (S. oleracea) ferredoxin:NADP⁺ reductase (SoFNR) + spinach ferredoxin (SoFd); lanes 3 and 4, EhNO1+SoFd; lanes 5 and 6, EhNO2+SoFd; lanes 7 and 8, EhNO1+EhFd1; lanes 9 and 10, EhNO2+EhFd1. Protein bands corresponding to the cross-linked proteins are indicated by an asterisk. The positions of the purified proteins are indicated on the right side of the gel. B, shown is an immunoblot analysis of the cross-linked samples using an anti-His antibody.

FIGURE 5.

Changes in the sensitivity of E. histolytica to metronidazole by overexpression of EhNOs. A, shown is an immunoblot analysis of EhNOs in the transformants expressing Myc-tagged EhNO1 and -2. Approximately 40 μg of total lysate from the pKT-MR (control), EhNO1 (pKT-MR-NO1), and EhNO2 (pKT-MR-NO2)-overexpressing transformants was electrophoresed on a SDS-PAGE gel under reducing conditions and subjected to immunoblot analysis using anti-EhNO1, anti-EhNO2, anti-Myc, and anti-EhCS1 (control) antibodies. Black and white arrows indicate endogenous and Myc-tagged EhNO1 and -2, respectively. B, susceptibility of transformed trophozoites to metronidazole is shown. Trophozoites (10⁴ cells/ml) were cultivated in the presence of 0–16 μm metronidazole for 48 h, and the number of viable cells was then counted. The percentages of living cells are shown relative to those of unexposed control cells. Error bars represent the S.E. of five independent experiments.

FIGURE 6.

Proposed in vivo reactions catalyzed by EhNO1 and -2. PFOR, pyruvate:ferredoxin oxidoreductase; Fd-red and Fd-oxi, reduced and oxidized form of ferredoxin; Prx, peroxiredoxin; Rbr, rubrerythrin; RNO₂/RNO₂^°, metronidazole/reduced metronidazole.