Abstract
Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through D-galacturonate/L-galactonate as representative intermediates. 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. The purified recombinant aldonolactonase expressed in E. 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 mM 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. 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.
