A key role for old yellow enzyme in the metabolism of drugs by Trypanosoma cruzi

Abstract

Trypanosoma cruzi is the etiological agent of Chagas' disease. So far, first choice anti-chagasic drugs in use have been shown to have undesirable side effects in addition to the emergence of parasite resistance and the lack of prospect for vaccine against T. cruzi infection. Thus, the isolation and characterization of molecules essential in parasite metabolism of the anti-chagasic drugs are fundamental for the development of new strategies for rational drug design and/or the improvement of the current chemotherapy. While searching for a prostaglandin (PG) F(2alpha) synthase homologue, we have identified a novel "old yellow enzyme" from T. cruzi (TcOYE), cloned its cDNA, and overexpressed the recombinant enzyme. Here, we show that TcOYE reduced 9,11-endoperoxide PGH(2) to PGF(2alpha) as well as a variety of trypanocidal drugs. By electron spin resonance experiments, we found that TcOYE specifically catalyzed one-electron reduction of menadione and beta-lapachone to semiquinone-free radicals with concomitant generation of superoxide radical anions, while catalyzing solely the two-electron reduction of nifurtimox and 4-nitroquinoline-N-oxide drugs without free radical production. Interestingly, immunoprecipitation experiments revealed that anti-TcOYE polyclonal antibody abolished major reductase activities of the lysates toward these drugs, identifying TcOYE as a key drug-metabolizing enzyme by which quinone drugs have their mechanism of action.

Figure 1.

PG production by T. cruzi epimastigote lysates. Stationary growth phase epimastigotes were cultured without the addition of AA into the culture medium, whereas lysates from these cells were incubated with or without 1 mM AA. Gray, black, and white bars indicate PGD₂, PGE₂, and PGF_2α, respectively. PG detection limits were less than 7.8, 7.8, and 3.6 pg/assay for PGD₂, PGE₂, and PGF_2α, respectively. Values shown are the mean from three independent experiments along with SE.

Figure 2.

(A) Reduction of PGH₂ by native TcOYE. Lane 1, substrate incubated in the absence of enzyme; lane 2, with 2 μg pure TcOYE; lane 3, with 20 μg heat-inactivated TcOYE. (B) SDS-PAGE of native TcOYE. 2 μg protein was resolved on a 14% SDS polyacrylamide gel and detected by Coomassie Brilliant Blue staining. Lane 1: molecular weight markers; lane 2, pure TcOYE. (C) Absorbance spectrum of 4 μg/μl oxidized TcOYE in 0.1 M sodium phosphate buffer, pH 7.0, at 37°C. (D) Partial amino acid sequences of the three internal fragments from the in-gel digestion of TcOYE.

Figure 3.

Multiple sequence alignment of deduced TcOYE amino acid sequence with representative members of the flavin-dependent oxidoreductases family. The amino acid sequences were taken from the public database. TcOYE is aligned with: LmMR, L. major morphine reductase (sequence data are available from GenBank/EMBL/DDBJ under accession no. AL390114); Agroba, Agrobacter tumefaciens oxidoreductase (accession no. NP_535816); Methorizo, Mesorhizobium loti morphinone reductase (accession no. NP_103610); Ec, E. coli N-ethylmaleimide reductase (accession no. NP_416167); Paer, P. aeruginosa xenobiotic reductase (accession no. NP_253046); Avinel, Azotobacter vinelandi 2-cyclohexane-one reductase (accession no. AB025798); Tomato, Lycopersicon esculentum 12-oxo phytodienoate reductase (accession no. AJ242551); Sc, Saccharomyces cerevisiae NADPH dehydrogenase 1 (accession no. Q02899); Candida, C. albicans NADPH dehydrogenase estrogen binding protein (accession no. P43084). * and : indicate identical amino acids and conserved amino acid substitutions, respectively. Boxes indicate the peptide sequences identified from purified native TcOYE.

Figure 4.

(A) Purification of recombinant TcOYE. TcOYE was expressed as a fusion protein with GSH in E. coli BL21. Lane 1, molecular weight markers; lane 2, lysates from noninduced E. coli BL21 cells expressing pGEX-TcOYE; lane 3, lysates from IPTG-induced E. coli BL21 cells expressing pGEX-TcOYE; lane 4, after affinity chromatography; lane 5, pure recombinant TcOYE. (B) Immunoblot analyses of cell extracts from T. cruzi epimastigote and T. brucei bloodstream form. After blotting, the membranes were incubated with anti-TcOYE or anti-TbPGFS polyclonal antibodies. On the left, SDS-PAGE of the lysates corresponding to T. brucei (Tb) and T. cruzi (Tc) is shown. The proteins were detected with Coomassie Brilliant Blue (CBB). The middle shows a Western blot for proteins incubated with anti-TcOYE polyclonal antibody. The right shows a Western blot for proteins incubated with anti-TbPGFS polyclonal antibody. (C) LC-MS chromatograms of the relative ion intensity for TcOYE-generated PGF_2α. Top, standard PGF_2α; left, substrate (9,11-endoperoxide PGH₂) showing traces of nonenzymatic degradation products (PGE₂, 11β-PGE₂, PGD₂, and PGJ₂); bottom, PGF_2α resulting from the reduction of the substrate by TcOYE. The chromatograms for PGE₂, 11β-PGE₂, PGD₂, and PGH₂ were visualized at m/z = 351.2, whereas that of PGF_2α was monitored at m/z = 353.2. (D) Mass spectra showing the [M-H]⁻ ions of authentic PGF_2α (top), PGH₂ (middle), and TcOYE-generated PGF_2α (bottom).

Figure 5.

Semiquinone and superoxide radical anion spectra obtained from menadione and β-lapachone. On the top, ESR spectrum of the semiquinone radical anion generated by the action of TcOYE on menadione after anaerobic incubation is shown. The bottom shows a computer simulation spectrum of the semiquinone radical anion with a g value of 2.0044 and hyperfine coupling constants a(4 H) = 3.0 G, a(3 H) = 0.66 G, and a maximum slope of line width ΔH _msl = 0.15 G. (B) On the top, ESR spectrum of the semiquinone radical anion generated by the action of TcOYE on β-lapachone after anaerobic incubation is shown. The bottom shows a computer simulation spectrum of the semiquinone radical anion with a g value of 2.0046 and hyperfine coupling constants a(1 H) = 3.3 G, a(2 H) = 1.7 G, and ΔH _msl = 0.7 G. (C) ESR spectrum of O₂ ^.- g _// = 2.09 and g _⊥ = 2.005 formed in the reaction of semiquinone radical anion with O₂ in 9 mM Tris/Cl, pH 7.4, at −155°C. g _// component is magnified five times.

Figure 6.

(A) Trypanocidal drug dose-dependence inhibition of 9,11-endoperoxide PGH₂ reduction by TcOYE. The enzymatic activity of the control (0 μM drug) was given a 100% value to calculate the percentage of residual activity. ♦, menadione; ▪, β-lapachone; ▴, nifurtimox; •, 4-nitroquinoline-N-oxide. Data are expressed as the mean along with SE from three independent experiments.

Figure 7.

(A) Immunoblot analysis of the supernatant from T. cruzi epimastigote cell lysates after immunoprecipitation with anti-TcOYE polyclonal antibody. Lane 1, lysates incubated without primary antibody; lane 2, anti-TbPGFS polyclonal antibody–treated lysates; lane 3, anti-TcOYE polyclonal antibody–treated lysates. After immunoblotting, the membrane was incubated with anti-TcOYE polyclonal antibody. (B) Supernatants from immunoprecipitated lysates were tested for their 9,11-endoperoxide PGH₂ reductase activity. Lanes 1–3 correspond to those in A.