Publications

• Characterization of Two <i>Arabidopsis</i> L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis.

  Aboobucker S.I., Suza W.P., Lorence A.

  L-Ascorbic acid (AsA, vitamin C) is an essential antioxidant for plants and animals. There are four known ascorbate biosynthetic pathways in plants: the L-galactose, L-gulose, D-galacturonate, and <i>myo</i>-inositol routes. These pathways converge into two AsA precursors: L-galactono-1,4-lactone ... >> More

  L-Ascorbic acid (AsA, vitamin C) is an essential antioxidant for plants and animals. There are four known ascorbate biosynthetic pathways in plants: the L-galactose, L-gulose, D-galacturonate, and <i>myo</i>-inositol routes. These pathways converge into two AsA precursors: L-galactono-1,4-lactone and L-gulono-1,4-lactone (L-GulL). This work focuses on the study of L-gulono-1,4-lactone oxidase (GulLO), the enzyme that works at the intersect of the gulose and inositol pathways. Previous studies have shown that feeding L-gulono-1,4-lactone to multiple plants leads to increased AsA. There are also reports showing GulLO activity in plants. We describe the first detailed characterization of a plant enzyme specific to oxidize L-GulL to AsA. We successfully purified a recombinant <i>Arabidopsis</i> GulLO enzyme (called AtGulLO5) in a transient expression system. The biochemical properties of this enzyme are similar to the ones of bacterial isozymes in terms of substrate specificity, subcellular localization, use of flavin adenine dinucleotide (FAD) as electron acceptor, and specific activity. AtGulLO5 is an exclusive dehydrogenase with an absolute specificity for L-GulL as substrate thus differing from the existing plant L-galactono-1,4-lactone dehydrogenases and mammalian GulLOs. Feeding L-GulL to <i>N. benthamiana</i> leaves expressing <i>AtGulLO5</i> constructs led to increased foliar AsA content, but it was not different from that of controls, most likely due to the observed low catalytic efficiency of AtGulLO5. Similar results were also obtained with another member of the AtGulLO family (AtGulLO3) that appears to have a rapid protein turnover. We propose that AsA synthesis through L-GulL in plants is regulated at the post-transcriptional level by limiting GulLO enzyme availability. << Less

  React Oxyg Species (Apex) 4:389-417(2017) [PubMed] [EuropePMC]

• L-galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.

  Leferink N.G.H., van den Berg W.A.M., van Berkel W.J.H.

  l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabid ... >> More

  l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l-galactono-1,4-lactone (K(m) = 0.17 mm, k(cat) = 134 s(-1)), l-gulono-1,4-lactone (K(m) = 13.1 mm, k(cat) = 4.0 s(-1)) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen (k(ox) = 6 x 10(2) m(-1).s(-1)). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l-galactono-1,4-lactone and l-gulono-1,4-lactone and released their FAD cofactor more easily than wild-type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed. << Less

  FEBS J. 275:713-726(2008) [PubMed] [EuropePMC]

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

  Wolucka B.A., Communi D.

  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 a ... >> More

  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. << Less

  FEBS J. 273:4435-4445(2006) [PubMed] [EuropePMC]