The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase

Abstract

We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.

FIGURE 1.

The pathway of AsA biosynthesis in Euglena and plants. A radio-tracer experiment provides evidence for the biosynthesis of AsA via uronic acids in Euglena. The unnumbered reactions are uncertain; however, enzyme 4 catalyzes the conversion of d-GlcUA to l-GulA (21). The pathway from d-Man 1-P to l-GalL (Man/Gal pathway, reactions 8-12) mainly operates in higher plants. Enzymes catalyzing the numbered reactions are: 1, UDP-Glc dehydrogenase; 2, UDP-Glc-4-epimerase; 3, UDP- d-GalUA pyrophosphatase/phosphorylase; 4, d-GalUA reductase; 5, ALase; 6, l-GalL dehydrogenase; 7, l-GulL oxidase/dehydrogenase; 8, GDP-d-Man pyrophosphorylase (VTC1); 9, GDP-Man-3′,5′-epimerase; 10, GDP-l-Gal phosphorylase (VTC2/VTC5); 11, l-Gal 1-P phosphatase (VTC4); 12, l-Gal dehydrogenase.

FIGURE 2.

Sequence alignment of Euglena ALase with SMP30 from rat and human and Drp35 from Staphylococcus and Zymomonas. Conserved amino acid residues for both calcium binding of Staphylococcus Drp35 and zinc binding of Euglena ALase are highlighted in white letters with a black background. The accession numbers are: Euglena ALase (AB306917), rat SMP30 (X69021), human SMP30 (BC050371), Staphylococcus aureus Drp35 (AB030228), and Zymomonas mobilis (AE008692).

FIGURE 3.

Production of recombinant Euglena ALase. The soluble proteins were extracted from E. coli cells transformed with an empty vector pColdTF or the vector containing the Euglena ALase (pColdTF/EgALase) after isopropyl 1-thio-β-d-galactopyranoside induction. The recombinant His- and TF-tag protein was purified in a nickel-nitrilotriacetic acid column. HRV3C protease was used to detach the fused tag. The samples were separated by 12.5% SDS-PAGE and visualized with Coomassie Brilliant Blue. M, molecular marker; lane 1, soluble fraction of cell extract transformed with an empty pColdTF vector; lane 2, soluble fraction of cell extract transformed with pColdTF/EgALase; lane 3, elution fraction from nickel-nitrilotriacetic acid; lane 4, purified recombinant EgALase after treatment with HRV 3C protease.

FIGURE 4.

Effect of divalent metal ions on Euglena ALase activity. A, ALase activity of the purified recombinant enzyme was determined in the presence of 75 μm concentrations of various divalent metal ions. B, ALase activity was determined at the indicated concentrations of ZnCl₂. Values are expressed as the mean ± S.E. of three independent experiments.

FIGURE 5.

Structural comparison of Staphylococcus Drp35 and predicted model of Euglena ALase. A, superim-position of Staphylococcus Drp35 and Euglena ALase. The protein structure of Drp35 and ALase is shown in yellow-green and pale blue, respectively. B, Ca²⁺ binding center in Drp35 and prediction of Zn²⁺-binding residues in Euglena ALase. The residues in Drp35 and ALase are shown in yellow-green and pale blue, respectively.

FIGURE 6.

Effect of amino acid substitution on Euglena ALase activity. ALase activities of affinity-purified mutant enzymes were determined at the indicated concentrations of ZnCl₂. Values are expressed as the mean ± S.E. for three independent experiments. WT, wild type.

FIGURE 7.

Phenotypic characterization of ALase-silenced Euglena cells. A, growth phenotype of dsRNA-introduced cells in a medium supplemented with l-GalL. Cells were restored and grown for 1 week in the presence of 1 mm l-GalL after the introduction of dsRNAs for ALase. Cultures of representative cells grown in a medium supplemented with 1 mm l-GalL were photographed 1 week after the dsRNA was introduced. B, Northern blot analysis. Each lane was loaded with 10 μg of total RNA from 1-week-old wild type (WT) cells and dsRNA-introduced cells. The RNA blot was hybridized with a ³²P-labeled ALase cDNA probe. The bottom shows ethidium bromide-stained rRNA as a RNA-loading control.

FIGURE 8.

Effect of precursors on AsA formation in Euglena. Euglena cells were incubated with 5 mm concentrations of each compound under light (55 μmol m^-2 s^-1) at 25 °C for 6 h. Values are expressed as the mean ± S.E. for three independent experiments. Values that were significantly different between control and treated cells are indicated; ^a, p < 0.05; ^b, p < 0.01.

FIGURE 9.

AsA level and expression of ALase under light in dark-grown Euglena. The cells were illuminated at an intensity of 55 μmol m^-2 s^-1. Samples were taken out at the indicated times and used for the determination of AsA, the assay of ALase, and Northern hybridization. Values are expressed as the mean ± S.E. for three experiments. A, AsA level. B, ALase activity. C, Northern blot analysis. Ten micrograms of total RNA extracted from each sample was electrophoresed through a formaldehyde-containing agarose gel, capillary blotted onto a nylon membrane, and hybridized with ³²P-labeled cDNA of Euglena ALase. Ethidium bromide staining of the rRNA is shown for the equality of loading. Values that were significantly different between control and illuminated cells are indicated. ^a, p < 0.05; ^b, p < 0.01.