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

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 Km 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 pathway via D-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight in to the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.