Biosynthesis of D-arabinose in mycobacteria - a novel bacterial pathway with implications for antimycobacterial therapy.

Decaprenyl-phospho-arabinose (beta-D-arabinofuranosyl-1-O-monophosphodecaprenol), the only known donor of d-arabinose in bacteria, and its precursor, decaprenyl-phospho-ribose (beta-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 decaprenyl-phospho-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 acid-related 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 (omega,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 phosphate-related lipids. In the field of immunology, the renaissance in mycobacterial polyisoprenoid research has led to the identification of mimetic mannosyl-beta-1-phosphomycoketides of pathogenic mycobacteria as potent lipid antigens presented by CD1c proteins to human T cells.

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

The recent identification of the VTC2 enzyme (GDP-l-galactose: hexose 1-phosphate guanylyltransferase) that forms with the GDP-mannose 3'',5'' epimerase an energy-conserving hub for the production of GDP-hexoses and l-galactose 1-phosphate [Laing et al., Proc. Natl. Acad. Sci. USA 104, 2007, 9534-9539], is a major breakthrough in our understanding of the biosynthesis of l-ascorbic acid (vitamin C) in plants. The observation that the VTC2 enzyme can use glucose 1-phosphate and GDP-d-glucose as substrates, and the long-known existence of an enigmatic GDP-d-mannose 2''-epimerase activity, have led us to the proposal of an extended VTC2 cycle that links photosynthesis with the biosynthesis of vitamin C and the cell-wall metabolism in plants. An evolutionary scenario is discussed for the acquisition of genes of eubacterial origin for the de novo synthesis of l-ascorbic acid in green algae and plants.

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.

Structure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.

GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization of both the 3' and 5' positions of GDP-alpha-D-mannose to yield GDP-beta-L-galactose. Production of the C5' epimer of GDP-alpha-D-mannose, GDP-beta-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-alpha-D-mannose, GDP-beta-L-galactose, and a mixture of GDP-beta-L-gulose with GDP-beta-L-4-keto-gulose to resolutions varying from 2.0 to 1.4 A. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-beta-L-galactose and GDP-beta-L-gulose. The reaction proceeds by C4' oxidation of GDP-alpha-D-mannose followed by epimerization of the C5' position to give GDP-beta-L-4-keto-gulose. This intermediate is either reduced to give GDP-beta-L-gulose or the C3' position is epimerized to give GDP-beta-L-4-keto-galactose, then C4' is reduced to GDP-beta-L-galactose. 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. On the basis of the structure of the GDP-beta-L-gulose/GDP-beta-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 alpha to the keto function of a carbohydrate identifies key common features.

Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions.

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.

Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus.

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.

GDP-mannose 3',5'-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants.

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-L-gulose. 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.