title: 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.
authors.affiliation: Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan. ishikawa@life.shimane-u.ac.jp
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nishikawa
authors.forename: Youngshun
authors.initials: Y
authors.lastname: Gao
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shibata
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Yabuta
authors.forename: Takanori
authors.initials: T
authors.lastname: Maruta
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shigeoka
mesh_terms: D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005056:Euglena gracilis / Q000201:enzymology* / Q000235:genetics; D006603:Hexuronic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D020418:Algal Proteins; D006603:Hexuronic Acids; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013400:Sugar Acids; C012991:galactonic acid; C007819:galacturonic acid; D002265:Carboxylic Ester Hydrolases; C020579:gluconolactonase; D001205:Ascorbic Acid
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