Abstract
Radioisotope tracer studies indicate that the biosynthesis pathway proceeds via D-galacturonate (D-GalUA) as the intermediate in Euglena cells. To clarify the Euglena pathway in detail, we have purified D-GalUA reductase and L-galactono-1,4-lactone (L-GalL) dehydrogenase, and cloned a cDNA encoding aldonolactonase (ALase). The purified D-GalUA reductase utilized NADPH as an electrondonor with Km values of 3.79 ± 0.5 mM and 4.67 ± 0.6 mM for D-GalUA and D-glucuronate, respectively. The recombinant ALase catalyzed L-galactonate and L-gulonate with the apparent Km values 1.55 ± 0.3 mM and 4.55 ± 0.23 mM, respectively. On the other hand, L-GalL dehydrogenase purified from Euglena mitochondria showed much higher affinity for L-GalL (Km=0.21 mM) rather than L-GulL (Km=113.9 mM). Furthermore, introduction of double-strand RNA for the ALase to Euglena cells resulted in the cell growth arrest. These results clearly demonstrated that D-GalUA pathway is the major route for ascorbate biosynthesis in Euglena.
