Biochem. J. (2007) 407
(419–426) (Printed in Great Britain)
The terminal step in vitamin C biosynthesis in Trypanosoma cruzi is mediated by a FMN-dependent galactonolactone oxidase
Flora J. LOGAN, Martin C. TAYLOR, Shane R. WILKINSON, Harparkash KAUR and John M. KELLY1
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
Humans lack the ability to synthesize 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-g-lactone and D-arabinono-g-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. FMN is an essential cofactor for enzyme activity and binds to TcGAL non-covalently. In other flavoproteins, a histidine residue located within the N-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), localized within a C-terminal motif (HWXK) that is a feature of ascorbate-synthesizing enzymes, are necessary for the FMN association. The conserved lysine residue within this motif (Lys-450) is not required for cofactor binding, but its replacement by glycine renders the protein completely inactive.
Key words: ascorbate, flavin mononucleotide (FMN), galactonolactone oxidase, trypanosome, vitamin C.
Abbreviations used: ALO, arabinonolactone oxidase; APX, ascorbate-dependent peroxidase; CHEFE, contour-clamped homogeneous electric field electrophoresis; GAL, galactonolactone oxidase; GALDH, galactonolactone dehydrogenase; GLO, gulonolactone oxidase; GULDH, gulonolactone dehydrogenase; IPTG, isopropyl b-D-thiogalactoside; NEM, N-ethylmaleimide; Ni-NTA, Ni2+-nitrilotriacetic acid; pCMB, p-chloromercuribenzoate; Tb, Trypanosoma brucei; Tc, Trypanosoma cruzi; TCA, trichloroacetic acid.
1To whom correspondence should be addressed (email john.kelly@lshtm.ac.uk).
Received 7 June 2007/10 July 2007; accepted 13 July 2007
Published as BJ Immediate Publication 13 July 2007, doi:10.1042/BJ20070766
© The Authors Journal compilation © 2007 Biochemical Society
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