Multiple independent L-gulonolactone oxidase (GULO) gene losses and vitamin C synthesis reacquisition events in non-Deuterostomian animal species.

BACKGROUND: L-ascorbate (Vitamin C) is an important antioxidant and co-factor in eukaryotic cells, and in mammals it is indispensable for brain development and cognitive function. Vertebrates usually become L-ascorbate auxothrophs when the last enzyme of the synthetic pathway, an L-gulonolactone oxidase (GULO), is lost. Since Protostomes were until recently thought not to have a GULO gene, they were considered to be auxothrophs for Vitamin C. RESULTS: By performing phylogenetic analyses with tens of non-Bilateria and Protostomian genomes, it is shown, that a GULO gene is present in the non-Bilateria Placozoa, Myxozoa (here reported for the first time) and Anthozoa groups, and in Protostomians, in the Araneae family, the Gastropoda class, the Acari subclass (here reported for the first time), and the Priapulida, Annelida (here reported for the first time) and Brachiopoda phyla lineages. GULO is an old gene that predates the separation of Animals and Fungi, although it could be much older. We also show that within Protostomes, GULO has been lost multiple times in large taxonomic groups, namely the Pancrustacea, Nematoda, Platyhelminthes and Bivalvia groups, a pattern similar to that reported for Vertebrate species. Nevertheless, we show that Drosophila melanogaster seems to be capable of synthesizing L-ascorbate, likely through an alternative pathway, as recently reported for Caenorhabditis elegans. CONCLUSIONS: Non-Bilaterian and Protostomians seem to be able to synthesize Vitamin C either through the conventional animal pathway or an alternative pathway, but in this animal group, not being able to synthesize L-ascorbate seems to be the exception rather than the rule.

The contribution of Arabidopsis homologs of L-gulono-1,4-lactone oxidase to the biosynthesis of ascorbic acid.

To clarify the involvement of seven Arabidopsis homologs of rat L-gulono-1,4-lactone (L-GulL) oxidase, AtGulLOs, in the biosynthesis of L-ascorbic acid (AsA), transgenic tobacco cells overexpressing the various AtGulLOs were generated. Under treatment with L-GulL, the levels of total AsA in three transgenic tobacco cell lines, overexpressing AtGulLO2, 3, or 5, were significantly increased as compared with those in control cells.

Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L-gulono-1,4-lactone dehydrogenase.

The last step of the biosynthesis of L-ascorbic acid (vitamin C) in plants and animals is catalyzed by L-gulono-1,4-lactone oxidoreductases, which use both L-gulono-1,4-lactone and L-galactono-1,4-lactone as substrates. L-gulono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient animals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis. A blast search using the rat L-gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited number of bacterial species, including several pathogens of human lungs, such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepacia and Bacillus anthracis. The genome of M. tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat L-gulono-1,4-lactone oxidase protein. The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized. The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes L-gulono-1,4-lactone (Km 5.5 mm) but not L-galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (Km 4.7 microm) and phenazine methosulfate as exogenous electron acceptors. Molecular oxygen does not serve as a substrate for the Rv1771 protein. Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain. In conclusion, M. tuberculosis produces a novel, highly specific L-gulono-1,4-lactone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C.

Leishmania donovani encodes a functional enzyme involved in vitamin C biosynthesis: arabino-1,4-lactone oxidase.

Plants and most animals can synthesize ascorbate (vitamin C) for their own requirements, but humans have lost this ability during evolution. The last step in the biosynthesis of L-ascorbic acid involves the conversion of an aldonolactone substrate to ascorbate (or analogues), reactions catalyzed by a family of flavoprotein aldonolactone oxidase/dehydrogenases. We report cloning, molecular characterization, localization and functional importance of arabinonolactone oxidase (LdALO), an enzyme from L. donovani, a protozoan parasite that causes visceral leishmaniasis. L. donovani arabinonolactone oxidase gene is 1509-bp and encodes a putative 502-amino acid protein with a molecular mass of 57-kDa. A 57-kDa protein was obtained by heterologous expression of LdALO in Escherichia coli. Recombinant arabinonolactone oxidase (LdALO) obeys Michaelis-Menten kinetics utilizing D-arabinono-γ-lactone as a substrate, a property characteristic of the yeast enzyme. Activity towards the mammalian substrate, L-gulono-γ-lactone, could not be detected. The inhibitor study profile suggested the essentiality of cysteine residues for the activity of this enzyme. LdALO displayed glycosomal localization as in other kinetoplastids. Overexpression of LdALO in L. donovani resulted in better ability of survival of the parasite within the host in comparison to the vector transfectants. D-arabinono-γ-lactone oxidase required for synthesizing ascorbate in Leishmania could be considered as a therapeutically exploitable target.