Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

Characterization of L-arabinose/D-galactose 1-dehydrogenase from Thermotoga maritima and its application in galactonate production.

The first hyperthermophilic L-arabinose/D-galactose 1-dehydrogenase (TmAraDH) from Thermotoga maritima was heterologously purified from Escherichia coli. It belongs to the Gfo/Idh/MocA protein family, prefers NAD+/NADP+ as a cofactor. The purified TmAraDH exhibited maximum activity toward L-arabinose at 75 °C and pH 8.0, and retained 63.7% of its activity after 24 h at 60 °C, and over 60% of its activity after holding a pH ranging from 7.0 to 9.0 for 1 h. Among all tested substrates, TmAraDH exclusively catalyzed the NAD(P)+-dependent oxidation of L-arabinose, D-galactose and D-fucose. The catalytic efficiency (kcat/Km) towards L-arabinose and D-galactose was 123.85, 179.26 min-1 mM-1 for NAD+, and 56.06, 18.19 min-1 mM-1 for NADP+, respectively. TmAraDH exhibited complete oxidative conversion in 12 h at 70 °C to D-galactonate with 5 mM D-galactose. Modelling provides structural insights into the cofactor and substrate recognition specificity. Our results suggest that TmAraDH have great potential for the conversion of L-arabinose and D-galactose to L-arabonate and D-galactonate.

Structural Characterization of L-Galactose Dehydrogenase: An Essential Enzyme for Vitamin C Biosynthesis.

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (ß/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

3,6-Anhydro-L-Galactose Dehydrogenase VvAHGD is a Member of a New Aldehyde Dehydrogenase Family and Catalyzes by a Novel Mechanism with Conformational Switch of Two Catalytic Residues Cysteine 282 and Glutamate 248.

3,6-anhydro-α-L-galactose (L-AHG) is one of the main monosaccharide constituents of red macroalgae. In the recently discovered bacterial L-AHG catabolic pathway, L-AHG is first oxidized by a NAD(P)+-dependent dehydrogenase (AHGD), which is a key step of this pathway. However, the catalytic mechanism(s) of AHGDs is still unclear. Here, we identified and characterized an AHGD from marine bacterium Vibrio variabilis JCM 19239 (VvAHGD). The NADP+-dependent VvAHGD could efficiently oxidize L-AHG. Phylogenetic analysis suggested that VvAHGD and its homologs represent a new aldehyde dehydrogenase (ALDH) family with different substrate preferences from reported ALDH families, named the L-AHGDH family. To explain the catalytic mechanism of VvAHGD, we solved the structures of VvAHGD in the apo form and complex with NADP+ and modeled its structure with L-AHG. Based on structural, mutational, and biochemical analyses, the cofactor channel and the substrate channel of VvAHGD are identified, and the key residues involved in the binding of NADP+ and L-AHG and the catalysis are revealed. VvAHGD performs catalysis by controlling the consecutive connection and interruption of the cofactor channel and the substrate channel via the conformational changes of its two catalytic residues Cys282 and Glu248. Comparative analyses of structures and enzyme kinetics revealed that differences in the substrate channels (in shape, size, electrostatic surface, and residue composition) lead to the different substrate preferences of VvAHGD from other ALDHs. This study on VvAHGD sheds light on the diversified catalytic mechanisms and evolution of NAD(P)+-dependent ALDHs.

Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.

The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of ß-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD'-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery.

Exopolysaccharide production in response to medium acidification is correlated with an increase in competition for nodule occupancy.

Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously shown to be more competitive for nodule occupancy. In this work, we show that strains carrying this mutation have galactose-dependent exopolysaccharide (EPS) phenotypes that were manifested as aberrant Calcofluor staining as well as decreased mucoidy when in an expR(+) genetic background. The aberrant Calcofluor staining was correlated with changes in the pH of the growth medium. Strains carrying dgoK mutations were subsequently demonstrated to show earlier acidification of their growth medium that was correlated with an increase expression of genes associated with succinoglycan biosynthesis as well as increased accumulation of high and low molecular weight EPS in the medium. In addition, it was shown that the acidification of the medium was dependent on the inability of S. meliloti strains to initiate the catabolism of galactose. To more fully understand why strains carrying the dgoK allele were more competitive for nodule occupancy, early nodulation phenotypes were investigated. It was found that strains carrying the dgoK allele had a faster rate of nodulation. In addition, nodule competition experiments using genetic backgrounds unable to synthesize either succinoglycan or EPSII were consistent with the hypothesis that the increased competition phenotype was dependent upon the synthesis of succinoglycan. Fluorescent microscopy experiments on infected root-hair cells, using the acidotropic dye Lysotracker Red DND-99, provide evidence that the colonized curled root hair is an acidic compartment.

L-arabinose/D-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of L-arabinose to L-arabonate with Saccharomyces cerevisiae.

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for L-arabonate production from L-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a D-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a L-arabinose/D-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP(+) but uses also NAD(+) as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards L-arabinose, D-galactose and D-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of L-arabinose, and the stable oxidation product detected is L-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear L-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for L-arabinose uptake, resulted in production of 18 g of L-arabonate per litre, at a rate of 248 mg of L-arabonate per litre per hour, with 86 % of the provided L-arabinose converted to L-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for L-arabonate production in yeast.

l-Galactose metabolism in Bacteroides vulgatus from the human gut microbiota.

A previously unknown metabolic pathway for the utilization of l-galactose was discovered in a prevalent gut bacterium, Bacteroides vulgatus. The new pathway consists of three previously uncharacterized enzymes that were found to be responsible for the conversion of l-galactose to d-tagaturonate. Bvu0219 (l-galactose dehydrogenase) was determined to oxidize l-galactose to l-galactono-1,5-lactone with kcat and kcat/Km values of 21 s(-1) and 2.0 × 10(5) M(-1) s(-1), respectively. The kinetic product of Bvu0219 is rapidly converted nonenzymatically to the thermodynamically more stable l-galactono-1,4-lactone. Bvu0220 (l-galactono-1,5-lactonase) hydrolyzes both the kinetic and thermodynamic products of Bvu0219 to l-galactonate. However, l-galactono-1,5-lactone is estimated to be hydrolyzed 300-fold faster than its thermodynamically more stable counterpart, l-galactono-1,4-lactone. In the final step of this pathway, Bvu0222 (l-galactonate dehydrogenase) oxidizes l-galactonate to d-tagaturonate with kcat and kcat/Km values of 0.6 s(-1) and 1.7 × 10(4) M(-1) s(-1), respectively. In the reverse direction, d-tagaturonate is reduced to l-galactonate with values of kcat and kcat/Km of 90 s(-1) and 1.6 × 10(5) M(-1) s(-1), respectively. d-Tagaturonate is subsequently converted to d-glyceraldehyde and pyruvate through enzymes encoded within the degradation pathway for d-glucuronate and d-galacturonate.

Expression, crystallization and preliminary X-ray analysis of rice L-galactose dehydrogenase.

In plants, L-galactose dehydrogenase (L-GalDH) is a key enzyme in the biosynthesis of ascorbic acid (AsA), which is well known as a unique antioxidant compound and a cofactor for many enzymes. L-GalDH catalyses the oxidation of L-galactose to L-galactono-1,4-lactone. Rice L-GalDH was overexpressed in Escherichia coli, purified and crystallized. Diffraction-quality rod-shaped crystals were grown using a sitting-drop vapour-diffusion method. The L-GalDH crystals exhibited the symmetry of space group P21 and diffracted to a resolution of 1.2 Å. The crystals had unit-cell parameters a = 46.8, b = 54.9, c = 56.9 Å, ß = 102.3°. On the basis of the Matthews coefficient (VM = 2.1 Å(3) Da(-1), solvent content of 42.3%), it was estimated that one peptide was present in the asymmetric unit.

Expression profiling of genes involved in ascorbate biosynthesis and recycling during fleshy root development in radish.

Ascorbate is a primary antioxidant and an essential enzyme cofactor in plants, which has an important effect on the development of plant root system. To investigate the molecular mechanisms of ascorbate accumulation during root development and reveal the key genes of the ascorbate biosynthesis and recycling pathways, the expression of 16 related genes together with ascorbate abundance were analyzed in the flesh and skin of radish (Raphanus sativus L.) fleshy root. The content of ascorbate decreased with root growth in both the flesh and skin. Expression of GDP-d-mannose pyrophosphorylase, GDP-d-mannose-3',5'-epimerase and d-galacturonate reductase were also decreased and correlated with ascorbate levels in the flesh. In the skin, the expression of GDP-d-mannose pyrophosphorylase and l-galactose dehydrogenase was correlated with ascorbate levels. These results suggested that ascorbate accumulation is affected mainly by biosynthesis rather than recycling in radish root, and the l-galactose pathway may be the major biosynthetic route of ascorbate, and moreover, the salvage pathway may also contribute to ascorbate accumulation. The data suggested that GDP-d-mannose pyrophosphorylase could play an important role in the regulation of ascorbate accumulation during radish fleshy taproot development.

Copper-induced synthesis of ascorbate, glutathione and phytochelatins in the marine alga Ulva compressa (Chlorophyta).

In order to analyze the synthesis of antioxidant and heavy metal-chelating compounds in response to copper stress, the marine alga Ulva compressa (Chlorophyta) was exposed to 10 µM copper for 7 days and treated with inhibitors of ASC synthesis, lycorine, and GSH synthesis, buthionine sulfoximine (BSO). The levels of ascorbate, in its reduced (ASC) and oxidized (DHA) forms, glutathione, in its reduced (GSH) and oxidized (GSSG) forms, and phytochelatins (PCs) were determined as well as activities of enzymes involved in ASC synthesis, L-galactose dehydrogenase (GDH) and L-galactono 1,4 lactone dehydrogenase (GLDH), and in GSH synthesis, γ-glutamylcysteine synthase (γ-GCS) and glutathione synthase (GS). The level of ASC rapidly decreased to reach a minimum at day 1 that remained low until day 7, DHA decreased until day 1 but slowly increased up to day 7 and its accumulation was inhibited by lycorine. In addition, GSH level increased to reach a maximal level at day 5 and GSSG increased up to day 7 and their accumulation was inhibited by BSO. Activities of GDH and GLDH increased until day 7 and GLDH was inhibited by lycorine. Moreover, activities of γ-GCS and GS increased until day 7 and γ-GCS was inhibited by BSO. Furthermore, PC2, PC3 and PC4, increased until day 7 and their accumulation was inhibited by BSO. Thus, copper induced the synthesis of ascorbate, glutathione and PCs in U. compressa suggesting that these compounds are involved in copper tolerance. Interestingly, U. compressa is, until now, the only ulvophyte showing ASC, GSH and PCs synthesis in response to copper excess.

Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages.

Ascorbic acid (AsA), as a unique antioxidant and enzyme cofactor, has multiple roles in plants. However, there is very limited information on the mechanism of AsA accumulation and controlling in leaves. In this study, we determined AsA accumulation levels, analyzed expression patterns of the genes involved in synthesizing via l-galactose pathway and recycling as well as enzyme activities in apple (Malus domestica Borkh) leaves with different age. AsA content was found to increase with leaf development, reaching the highest level in 20-day-old leaves. This level was maintained in mature leaves until the dropping in senescent leaves. Comparing with young and senescent leaves, mature leaves had higher capability for AsA synthesis with high expression levels and activity of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase. The mRNA expression of genes involved in AsA synthesis also showed highest abundance in 20-day-old leaves, though GDP-mannose-3',5'-epimerase and l-galactose-1-phosphate phosphatase expression reached the highest levels before 20 days old. These results suggest that AsA accumulation in apple leaves mainly occurs during the transition phase from young to mature leaves with high rates of synthesis and recycling, and that l-galactose-1-phosphate phosphatase could play an important role in regulating AsA biosynthesis via the l-galactose pathway.

Influence of light on ascorbate formation and metabolism in apple fruits.

To further understand the regulatory mechanism of light on the formation of ascorbic acid (AsA) in the sink organs of plants, a systematical investigation on AsA levels, activities of two key biosynthsis enzymes and their mRNA expression as well as the recycling was performed in the fruits of apple (Malus domestica Borkh), under different levels of shade. After the whole trees were shaded with the sun-light about 50-55% for 20 days, AsA levels were significantly decreased in fruit peel, flesh and leaves, while mRNA expression levels and activities of L-galactose dehydrogenase (L-GalDH, EC 1.1.1.117) and L-galactono-1,4-lactone dehydrogenase (L-GalLDH, EC 1.3.2.3) as well as activities of recycling enzymes was clearly declined in the leaf and peel but not in the flesh. By shading fruits only for 20 days, AsA levels, relative mRNA levels and activities of L-GalDH and L-GalLDH as well as activities of recycling enzymes all showed obvious decrease in the peel, but not in the flesh. However, their levels in the peel were markedly increased after the full shade was removed and re-exposed these fruits on natural light for 5 days. It is concluded that light affects AsA biosynthesis and recycling in the peel and leaf, but did not in the fresh. Results also suggest that apple fruit is potential to biosynthesize AsA via the L-galactose pathway, and AsA content in the fruits may depend partly on levels of AsA or other photochemistry controlled by light in the leaves.

Environmental stresses induce health-promoting phytochemicals in lettuce.

Plants typically respond to environmental stresses by inducing antioxidants as a defense mechanism. As a number of these are also phytochemicals with health-promoting qualities in the human diet, we have used mild environmental stresses to enhance the phytochemical content of lettuce, a common leafy vegetable. Five-week-old lettuce (Lactuca sativa L.) plants grown in growth chambers were exposed to mild stresses such as heat shock (40 degrees C for 10 min), chilling (4 degrees C for 1d) or high light intensity (800 micromolm(-2)s(-1) for 1d). In response to these stresses, there was a two to threefold increase in the total phenolic content and a significant increase in the antioxidant capacity. The concentrations of two major phenolic compounds in lettuce, chicoric acid and chlorogenic acid, increased significantly in response to all the stresses. Quercetin-3-O-glucoside and luteolin-7-O-glucoside were not detected in the control plants, but showed marked accumulations following the stress treatments. The results suggest that certain phenolic compounds can be induced in lettuce by environmental stresses. Of all the stress treatments, high light produced the greatest accumulation of phenolic compounds, especially following the stress treatments during the recovery. In addition, key genes such as phenylalanine ammonia-lyase (PAL), l-galactose dehydrogenase (l-GalDH), and gamma-tocopherol methyltransferase (gamma-TMT) involved in the biosynthesis of phenolic compounds, ascorbic acid, and alpha-tocopherol, respectively, were rapidly activated by chilling stress while heat shock and high light did not appear to have an effect on the expression of PAL and gamma-TMT. However, l-GalDH was consistently activated in response to all the stresses. The results also show that these mild environmental stresses had no adverse effects on the overall growth of lettuce, suggesting that it is possible to use mild environmental stresses to successfully improve the phytochemical content and hence the health-promoting quality of lettuce with little or no adverse effect on its growth or yield.

[Relationship between ascorbic acid accumulation and related enzyme activities in fruit of Rosa roxburghii Tratt].

Ascorbic acid (AsA) and sugar levels, together with activities of L-galactono-1,4-lactone dehydrogenase (GalLDH, a key enzyme in AsA biosynthesis in higher plant), AAO, AAP, MDAR and DHAR in fruit of Rosa roxburghii Tratt were measured during development. The result showed that AsA accumulated continually but with a slow, fast and slow accumulating rate in the R. roxburghii fruit during its development, in which the period from the end of June to the early of August was the most important period of AsA accumulation, since the AsA accumulated in this stage accounting for approximately 90% of the final level (Fig.1B). Changes in GalLDH activity coincided with AsA accumulating rate during fruit development (Fig.2B). The extremely significant positive correlation existed between the GalLDH activity and AsA accumulating rate (r(2)=0.783**) (Fig.3). Transitory and low activities of AAO and AAP in the fruit were detected in the initial stage of development (Table 1), suggesting that little AsA degraded by the activities of the two oxidative enzymes during the whole development. This data elucidated, at least in part, the reason for high accumulation of AsA in the R. roxburghii fruit. Furthermore, no activity of MDAR or DHAR in the fruit was detected during the whole development, implying that the two enzymes are not key factors for contribution of AsA level. No correlation was found between AsA content and sugar contents in the R. roxburghii fruit.

Properties of the recombinant glucose/galactose dehydrogenase from the extreme thermoacidophile, Picrophilus torridus.

In Picrophilus torridus, a euryarchaeon that grows optimally at 60 degrees C and pH 0.7 and thus represents the most acidophilic thermophile known, glucose oxidation is the first proposed step of glucose catabolism via a nonphosphorylated variant of the Entner-Doudoroff pathway, as deduced from the recently completed genome sequence of this organism. The P. torridus gene for a glucose dehydrogenase was cloned and expressed in Escherichia coli, and the recombinant enzyme, GdhA, was purified and characterized. Based on its substrate and coenzyme specificity, physicochemical characteristics, and mobility during native PAGE, GdhA apparently resembles the main glucose dehydrogenase activity present in the crude extract of P. torridus DSM 9790 cells. The glucose dehydrogenase was partially purified from P. torridus cells and identified by MS to be identical with the recombinant GdhA. P. torridus GdhA preferred NADP+ over NAD+ as the coenzyme, but was nonspecific for the configuration at C-4 of the sugar substrate, oxidizing both glucose and its epimer galactose (Km values 10.0 and 4.5 mM, respectively). Detection of a dual-specific glucose/galactose dehydrogenase points to the possibility that a 'promiscuous' Entner-Doudoroff pathway may operate in P. torridus, similar to the one recently postulated for the crenarchaeon Sulfolobus solfataricus. Based on Zn2+ supplementation and chelation experiments, the P. torridus GdhA appears to contain structurally important zinc, and conserved metal-binding residues suggest that the enzyme also contains a zinc ion near the catalytic site, similar to the glucose dehydrogenase enzymes from yeast and Thermoplasma acidophilum. Strikingly, NADPH, one of the products of the GdhA reaction, is unstable under the conditions thought to prevail in Picrophilus cells, which have been reported to maintain the lowest cytoplasmic pH known (pH 4.6). At the optimum growth temperature for P. torridus, 60 degrees C, the half-life of NADPH at pH 4.6 was merely 2.4 min, and only 1.7 min at 65 degrees C (maximum growth temperature). This finding suggests a rapid turnover of NADPH in Picrophilus.

Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii.

Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter(-1), converting 40% (wt/vol) of the starting compound L-galactose.

Designed chimaeric galactosyl-mimodye ligands for the purification of Pseudomonas fluorescens beta-galactose dehydrogenase.

Two chimaeric galactosyl-mimodye ligands were designed and applied to the purification of Pseudomonas fluorescens galactose dehydrogenase (GaDH). The chimaeric affinity ligands comprised a triazine ring on which were anchored: (i) an anthraquinone moiety that pseudomimics the adenine part of NAD+, (ii) a galactosyl-mimetic moiety (D-galactosamine for ligand BM1 or shikimate for ligand BM2), bearing an aliphatic 'linker', that mimics the natural substrate galactose, and (iii) a long hydrophilic 'spacer'. The mimodye-ligands were immobilised to 1,1-carbonyldiimidazole-activated agarose chromatography support, via the spacer's terminal amino-group, to produce the respective mimodye adsorbents. Both immobilized mimodyes successfully bound P. fluorescens GaDH but failed to bind the enzyme from rabbit muscle. Adsorbent BM1 bound GaDH from green peas and Baker's yeast, but adsorbent BM2 failed to do so. The mimodye-ligand comprising D(+)-galactosamine (BM1), compared to BM2, exhibited higher purifying ability and enzyme recovery for P. fluorescens GaDH. The dissociation constants (KD) of BM1 and BM2 for P. fluorescens GaDH were determined by analytical affinity chromatography to be 5.9 microM and 15.4 microM, respectively. The binding capacities of adsorbents BM1 and BM2 were 18 U/mg adsorbent and 6 U/mg adsorbent, respectively. Adsorbents BM1 and BM2 were integrated in two different protocols for the purification P. fluorescens GaDH. Both protocols comprised as a common first step DEAE anion-exchange chromatography, with a second step of affinity chromatography on BM1 or BM2, respectively. The purified GaDH obtained from the protocols using BM1 and BM2 showed specific activities equal to 1077 and 854 U/mg, respectively. The former is the highest reported so far and the enzyme appeared as a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Synthesis of L-ascorbic acid in the phloem.

BACKGROUND: Although plants are the main source of vitamin C in the human diet, we still have a limited understanding of how plants synthesise L-ascorbic acid (AsA) and what regulates its concentration in different plant tissues. In particular, the enormous variability in the vitamin C content of storage organs from different plants remains unexplained. Possible sources of AsA in plant storage organs include in situ synthesis and long-distance transport of AsA synthesised in other tissues via the phloem. In this paper we examine a third possibility, that of synthesis within the phloem. RESULTS: We provide evidence for the presence of AsA in the phloem sap of a wide range of crop species using aphid stylectomy and histochemical approaches. The activity of almost all the enzymes of the primary AsA biosynthetic pathway were detected in phloem-rich vascular exudates from Cucurbita pepo fruits and AsA biosynthesis was demonstrated in isolated phloem strands from Apium graveolens petioles incubated with a range of precursors (D-glucose, D-mannose, L-galactose and L-galactono-1,4-lactone). Phloem uptake of D-[U-14C]mannose and L-[1-14C]galactose (intermediates of the AsA biosynthetic pathway) as well as L-[1-14C]AsA and L-[1-14C]DHA, was observed in Nicotiana benthamiana leaf discs. CONCLUSIONS: We present the novel finding that active AsA biosynthesis occurs in the phloem. This process must now be considered in the context of mechanisms implicated in whole plant AsA distribution. This work should provoke studies aimed at elucidation of the in vivo substrates for phloem AsA biosynthesis and its contribution to AsA accumulation in plant storage organs.