The terminal step in vitamin C biosynthesis in Trypanosoma cruzi is mediated by a FMN-dependent galactonolactone oxidase
Résumé
Humans lack the ability to synthesise vitamin C (ascorbate), due to the absence of gulonolactone oxidase, the last enzyme in the biosynthetic pathway in most other mammals. The corresponding oxidoreductase in trypanosomes therefore represents a target that may be therapeutically exploitable. This is reinforced by our observation that Trypanosoma cruzi, the causative agent of Chagas disease, lacks the capacity to scavenge ascorbate from its environment and is therefore dependent on biosynthesis to maintain intracellular levels of this vitamin. Here, we show that T. cruzi galactonolactone oxidase (TcGAL) can utilize both L-galactono-γ-lactone and D-arabinono-γ-lactone as substrates for synthesis of vitamin C, in reactions that obey Michaelis-Menten kinetics. It is >20-fold more active than the analogous enzyme from the African trypanosome Trypanosoma brucei. Flavin mononucleotide (FMN) is an essential cofactor for enzyme activity and binds to TcGAL non-covalently. In other flavoproteins, a histidine residue located within the amino terminal flavin-binding motif has been shown to be crucial for cofactor binding. Using site-directed mutagenesis, we show that the corresponding residue in TcGAL (Lys-55) is not essential for this interaction. In contrast, we find that histidine and tryptophan residues (His-447 and Trp-448), localised within a carboxyl terminal motif (HWXK) that is a feature of ascorbate-synthesising enzymes, are necessary for the FMN-association. The conserved lysine residue within this motif (Lys-450) is not required for cofactor binding, but its substitution by glycine renders the protein completely inactive.
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