Functional Characterization ofD-Galacturonic Acid Reductase, a Key Enzyme of the Ascorbate Biosynthesis Pathway, fromEuglena gracilis

Abstract

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.

The Pathway via D-Galacturonate/L-Galactonate Is Significant for Ascorbate Biosynthesis in Euglena gracilis

“…On the other hand, Euglena ALase showed redox sensitivity caused by H 2 O 2 treatment (Table 3). Activation of D-GalUA reductase by H 2 O 2 has also been observed in our previous study (21). Because AsA levels in Euglena cells increased under oxidative stress (20,27) and light (Fig.…”

“…This result provides compelling evidence that ALase is essential for AsA biosynthesis, and the pathway via uronic acid is the major source of AsA in Euglena cells. We previously reported that a D-GalUA reductase purified from Euglena had almost the same catalytic efficiency with two uronic acids, D-GalUA and D-GlcUA (21). Judging from the enzymology parameters of both D-GalUA reductase and ALase, it is difficult to understand which is the more effective route to promote the level of AsA in Euglena cells.…”

“…D-GalL and D-glucono-␦-lactone were purchased from Wako Pure Chemical (Osaka, Japan). L-GalA and L-GulA were prepared by the hydrolysis of L-GalL and L-GulL, respectively, under basic conditions (21). Twenty milliliters of 0.3 M NaOH was added to 100 l of 10 mM L-GalL or L-GulL, the mixture was vigorously agitated by vortexing for 20 s, and 20 l of 0.3 M hydrochloric acid was added to neutralize the solution.…”

“…Gene in Euglena Cells-Our previous identification and characterization of D-GalUA reductase from Euglena indicated that the occurrence of ALase is essential for the conversion of L-GalA, the resultant product of D-GalUA reduction, to L-GalL in this organism (21). Therefore, a BLASTp search was performed against the Euglena EST data base of the Protest EST Program using the amino acid sequence of the rat gluconolactonase involved in the biosynthesis of AsA in mammals (6) as a query.…”

Recent Advances in Ascorbate Biosynthesis and the Physiological Significance of Ascorbate Peroxidase in Photosynthesizing Organisms

“…This pathway actually operates in the unicellular alga Euglena gracilis. 25,26) On the other hand, the product of the myo-inositol oxygenase reaction is presumably a D-glucuronate, assuming the occurrence of the AsA biosynthetic pathway in higher plants, similarly to animals. In the cases of both alternative L-gulose and D-glucuronate pathways, to generate AsA, the presence of L-gulono-1,4-lactone (L-GulL) oxidase or dehydrogenase is essential to complete them.…”