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

Abstract

Vitamin C (l-ascorbic acid; L-AA) is an important metabolite of plants and animals. It functions as an antioxidant (or pro-oxidant), an enzyme cofactor, an effector of gene expression, and a modulator of reactive oxygen species (ROS)-mediated cell signaling. L-AA is therefore involved in a wide array of crucial physiologic processes, including: biosynthesis of collagen and other hydroxyproline ⁄ hydroxylysine-containing proteins ⁄ peptides; synthesis of secondary metabolites, hormones and cytokines [1] Plants synthesize ascorbic acid via de novo and salvage pathways [7], whereas a de novo pathway involving UDP-d-glucuronic acid operates in animals [8]. l-Gulono-1,4-lactone is a direct precursor of vitamin C in animals [8], but also in plants [9] and in some protists [10]. In plants, L-AA can be formed additionally from l-galactono-1,4-lactone by a highly specific mitochondrial dehydrogenase (EC 1.3.2.3) [11,12] 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 (K m 5.5 mm) but not l-galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (K m 4.7 lm) 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.Abbreviations GST, glutathione-S-transferase; IPTG, isopropyl thio-b-D-galactoside; L-AA, L-ascorbic acid; MALDI Q-TOF, MALDI quadrupole TOF; ROS, reactive oxygen species.

Characterization of Two Arabidopsis L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis

“…The characteristic flavin spectrum was absent and the enzyme activity was the same with or without exogenous addition of 100 μM FAD (data not shown). This is similar to the findings with the L-GulL dehydrogenase (GulLDH) enzyme from Mycobacterium tuberculosis [14]. …”

The VTC2 cycle and the de novo biosynthesis pathways for vitamin C in plants: An opinion

“…The recent discovery of a functional bacterial gene for vitamin C synthesis, the L-gulono-1,4-lactone dehydrogenase of the Mycobacterium tuberculosis pathogen, and the fact that similar proteins are present in many bacterial pathogens and symbionts (Wolucka and Communi, 2006), further support the hypothesis of a eubacterial origin of vitamin C-related genes in the evolution of higher eukaryotes. A common ancestor of green algae and plants had assembled evolutionary novel VTC2 and L-gulono-1,4-lactone dehydrogenase genes, in addition to the more ancient algal GDP-mannose 3 00 ,5 00 -epimerase, thus forming a formidable enzymatic machinery for an extremely efficient production of L-ascorbic acid.…”

“…This observation provides indirect evidence for the involvement of the putative Lgluono-1,4-lactone dehydrogenases in the de novo synthesis of vitamin C. Clearly, L-galactonolactone dehydrogenase gene was retained and well-conserved in higher plants, but highly mutated or lost in green algae, probably after the acquisition of the L-gluono-1,4-lactone dehydrogenase gene. BLAST searches of eukaryotic and prokaryotic genomes revealed that only few species of bacteria contain a protein similar to the plant L-gulono-1,4-lactone dehydrogenases, and the closest homolog is present in a marine proteobacterium Hahella chejuensis (27% identity) that is closely associated with algal populations, and in some actinobacteria (Wolucka and Communi, 2006). It seems probable, therefore, that an ancient L-gulono-1,4-lactone dehydrogenase gene of a common ancestor of green algae and land plants could be acquired via horizontal gene transfer from an ancient marine eubacterium.…”

“…Surprisingly, the highest increase of vitamin C content (5-7 folds) was reported in plants overexpressing the rat gene for L-ascorbic acid synthesis, L-gulono-1,4-lactone oxidase (Jain and Nessler, 2000). Perhaps, using other non-plant genes to generate plants with higher vitamin C contents as, for example, bacterial L-gulono-1,4-lactone dehydrogenases (Wolucka and Communi, 2006), could be an option.…”

An Altered Mycobacterium tuberculosis Metabolome Induced by katG Mutations Resulting in Isoniazid Resistance

“…Furthermore, ascorbic acid is a well-known antioxidant, and elevated ascorbic acid oxidation to threonic acid, in various other organisms is usually indicative of increased oxidative stress (42). Although an ascorbic acid synthesis pathway has been previously described for M. tuberculosis (43), the oxidative breakdown product of this, threonic acid, most probably induced by elevated hydrogen peroxide, as detected in these isoniazid-resistant strains, has never been described for M. tuberculosis. This may be due to two possible reasons: first, the elevated hydrogen peroxide due to the mutation in katG, and second, the elevated ascorbic acid synthesis by the organism to cope with this, or both.…”