Despite its importance for agriculture, bioindustry, and nutrition, the fundamental process of L-ascorbic acid (vitamin C) biosynthesis in plants is not completely elucidated, and little is known about its regulation. The recently identified GDP-Man 3,5-epimerase catalyzes a reversible epimerization of GDP-D-mannose that precedes the committed step in the biosynthesis of vitamin C, resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl linkage. Here, we characterize the native and recombinant GDP-Man 3,5-epimerase of Arabidopsis thaliana. GDP and GDP-D-glucose are potent competitive inhibitors of the enzyme, whereas GDP-L-fucose gives a complex type of inhibition. The epimerase contains a modified version of the NAD binding motif and is inhibited by NAD(P)H and stimulated by NAD(P) ؉ . A feedback inhibition of vitamin C biosynthesis is observed apparently at the level of GDP-Man 3,5-epimerase. The epimerase catalyzes at least two distinct epimerization reactions and releases, besides the well known GDP-L-galactose, a novel intermediate: GDP-Lgulose. The yield of the epimerization varies and seems to depend on the molecular form of the enzyme. Both recombinant and native enzymes co-purified with a Hsp70 heat-shock protein (Escherichia coli DnaK and A. thaliana Hsc70.3, respectively). We speculate, therefore, that the Hsp70 molecular chaperones might be involved in folding and/or regulation of the epimerase. In summary, the plant epimerase undergoes a complex regulation and could control the carbon flux into the vitamin C pathway in response to the redox state of the cell, stress conditions, and GDP-sugar demand for the cell wall/glycoprotein biosynthesis. Exogenous L-gulose and L-gulono-1,4-lactone serve as direct precursors of L-ascorbic acid in plant cells. We propose an L-gulose pathway for the de novo biosynthesis of vitamin C in plants.Vitamin C (L-ascorbic acid (L-AA) 1 ) acts as an enzyme cofactor and an antioxidant. In plants it may represent one of the major soluble carbohydrates and is involved in crucial physiological processes such as biosynthesis of the cell wall, phytohormones, and secondary metabolites, cell division and growth, and stress resistance and photoprotection (for review, see Ref.1). Large variations in vitamin C content (from 0.003 to 1% of fresh weight; w/w), reported for different plant species, organs, and tissues (2), are intimately linked to the vitamin biosynthesis, stability, and function. Plants, algae, and the majority of animals are able to synthesize vitamin C. Humans, however, lack L-gulono-1,4-lactone oxidase, the last enzyme of the vitamin C pathway in animals, and require L-AA as an essential micronutrient. L-AA biosynthetic genes can be engineered to increase vitamin C content of plants (3-4), in view of improving the nutritional value and stress resistance of crops, but also potentially exploited for the industrial production of vitamin C (5).The biosynthesis of vitamin C in plants is not completely elucidated, and its regulation is largely unknown....

Decaprenyl-phospho-arabinose (b-d-arabinofuranosyl-1-O-monophosphodecaprenol), the only known donor of d-arabinose in bacteria, and its precursor, decaprenyl-phospho-ribose (b-d-ribofuranosyl-1-O-monophosphodecaprenol), were first described in 1992. En route to d-arabinofuranose, the decaprenyl-phospho-ribose 2¢-epimerase converts decaprenyl-phospho-ribose to decaprenyl-phospho-arabinose, which is a substrate for arabinosyltransferases in the synthesis of the cell-wall arabinogalactan and lipoarabinomannan polysaccharides of mycobacteria. The first step of the proposed decaprenylphospho-arabinose biosynthesis pathway in Mycobacterium tuberculosis and related actinobacteria is the formation of d-ribose 5-phosphate from sedoheptulose 7-phosphate, catalysed by the Rv1449 transketolase, and ⁄ or the isomerization of d-ribulose 5-phosphate, catalysed by the Rv2465 d-ribose 5-phosphate isomerase. d-Ribose 5-phosphate is a substrate for the Rv1017 phosphoribosyl pyrophosphate synthetase which forms 5-phosphoribosyl 1-pyrophosphate (PRPP). The activated 5-phosphoribofuranosyl residue of PRPP is transferred by the Rv3806 5-phosphoribosyltransferase to decaprenyl phosphate, thus forming 5¢-phosphoribosyl-monophospho-decaprenol. The dephosphorylation of 5¢-phosphoribosyl-monophospho-decaprenol to decaprenyl-phospho-ribose by the putative Rv3807 phospholipid phosphatase is the committed step of the pathway. A subsequent 2¢-epimerization of decaprenyl-phospho-ribose by the heteromeric Rv3790 ⁄ Rv3791 2¢-epimerase leads to the formation of the decaprenyl-phospho-arabinose precursor for the synthesis of the cell-wall arabinans in Actinomycetales. The mycobacterial 2¢-epimerase Rv3790 subunit is similar to the fungal d-arabinono-1,4-lactone oxidase, the last enzyme in the biosynthesis of d-erythroascorbic acid, thus pointing to an evolutionary link between the d-arabinofuranose-and l-ascorbic acidrelated pathways. Decaprenyl-phospho-arabinose has been a lead compound for the chemical synthesis of substrates for mycobacterial arabinosyltransferases and of new inhibitors and potential antituberculosis drugs. The peculiar (x,mono-E,octa-Z) configuration of decaprenol has yielded insights into lipid biosynthesis, and has led to the identification of the novel Z-polyprenyl diphosphate synthases of mycobacteria. Mass spectrometric methods were developed for the analysis of anomeric linkages and of dolichol phosphaterelated lipids. In the field of immunology, the renaissance in mycobacterial polyisoprenoid research has led to the identification of mimetic mannosyl-b-1-phosphomycoketides of pathogenic mycobacteria as potent lipid antigens presented by CD1c proteins to human T cells.Abbreviations ALO, D-arabinono-1,4-lactone oxidase; Araf, D-arabinofuranose; GLO, L-gulono-1,4-lactone oxidase; PRPP, 5-phosphoribosyl 1-pyrophosphate.

Vitamin C (L-ascorbic acid) is an important primary metabolite of plants that functions as an antioxidant, an enzyme cofactor, and a cell-signalling modulator in a wide array of crucial physiological processes, including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, and growth. Plants synthesize ascorbic acid via de novo and salvage pathways, but the regulation of its biosynthesis and the mechanisms behind ascorbate homeostasis are largely unknown. Jasmonic acid and its methyl ester (jasmonates) mediate plant responses to many biotic and abiotic stresses by triggering a transcriptional reprogramming that allows cells to cope with pathogens and stress. By using 14C-mannose radiolabelling combined with HPLC and transcript profiling analysis, it is shown that methyl jasmonate treatment increases the de novo synthesis of ascorbic acid in Arabidopsis and tobacco Bright Yellow-2 (BY-2) suspension cells. In BY-2 cells, this stimulation coincides with enhanced transcription of at least two late methyl jasmonate-responsive genes encoding enzymes for vitamin C biosynthesis: the GDP-mannose 3'',5''-epimerase and a putative L-gulono-1,4-lactone dehydrogenase/oxidase. As far as is known, this is the first report of a hormonal regulation of vitamin C biosynthesis in plants. Finally, the role of ascorbic acid in jasmonate-regulated stress responses is reviewed.

GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana catalyses the epimerization of both the 3′ and 5′ positions of GDP-α-D-mannose to yield GDP-β-L-galactose. Production of the C5′ epimer of GDP-α-D-mannose, GDP-β-L-gulose, has also been reported. The reaction occurs as part of vitamin C biosynthesis in plants. We have determined structures of complexes of GME with GDP-α-D-mannose, GDP-β-L-galactose and a mixture of GDP-β-L-gulose with GDP-β-L-4-keto-gulose, to resolutions varying from 2.0 Å to 1.4 Å. The enzyme has the classical extended short chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-β-L-galactose and GDP-β-L-gulose. The reaction proceeds by C4′ oxidation of GDP-α-D-mannose followed by epimerization of the C5′ position to give GDP-β-L-4-keto-gulose. This intermediate is either reduced to give GDP-β-L-gulose or the C3′ position is epimerized to give GDP-β-L-4-keto-galactose, then C4′ is reduced to GDP-β-Lgalactose. The combination of oxidation, epimerization and reduction in a single active site is unusual. Structural analysis coupled to site directed mutagenesis suggests C145 and K217 as the acid / base pair responsible for both epimerizations. Based on the structure of the GDP-β-L-gulose/ GDP-β-L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate is likely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract a protein α to the keto function of a carbohydrate identifies key common features.

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The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, of L-galactosyl residues, catalyzed by a largely unknown GDP-D-mannose 3؆,5؆-epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-D-mannose 3؆,5؆-epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a BLAST search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-D-mannose 5؆-epimerase. The plant GDP-D-mannose 3؆,5؆-epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase͞reductase family. The enzyme was cloned and expressed in Escherichia coli cells.

A nonpolar mutation was made in the oac2 gene of Azorhizobium caulinodans. oac2 is an ortholog of the Salmonella typhimurium rfbD gene that encodes a dTDP-L-rhamnose synthase. The knockout of oac2 changed the lipopolysaccharide (LPS) pattern and affected the extracellular polysaccharide production but had no effect on bacterial hydrophobicity. Upon hot phenol extraction, the wild-type LPS partitioned in the phenol phase. The LPS fraction of ORS571-oac2 partitioned in the water phase and had a reduced rhamnose content and truncated LPS molecules on the basis of faster migration in detergent gel electrophoresis. Strain ORS571-oac2 induced ineffective nodule-like structures on Sesbania rostrata. There was no clear demarcation between central and peripheral tissues, and neither leghemoglobin nor bacteroids were present. Light and electron microscopy revealed that the mutant bacteria were retained in enlarged, thick-walled infection threads. Infection centers emitted a blue autofluorescence under UV light. The data indicate that rhamnose synthesis is important for the production of surface carbohydrates that are required to sustain the compatible interaction between A. caulinodans and S. rostrata.

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.