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The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.
- Source :
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The Journal of biological chemistry [J Biol Chem] 2008 Nov 07; Vol. 283 (45), pp. 31133-41. Date of Electronic Publication: 2008 Sep 09. - Publication Year :
- 2008
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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.
- Subjects :
- Algal Proteins genetics
Amino Acid Sequence
Animals
Ascorbic Acid genetics
Carboxylic Ester Hydrolases genetics
Escherichia coli enzymology
Escherichia coli genetics
Euglena gracilis genetics
Molecular Sequence Data
Protozoan Proteins genetics
Rats
Recombinant Proteins genetics
Recombinant Proteins metabolism
Algal Proteins metabolism
Ascorbic Acid biosynthesis
Carboxylic Ester Hydrolases metabolism
Euglena gracilis enzymology
Hexuronic Acids metabolism
Protozoan Proteins metabolism
Sugar Acids metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 283
- Issue :
- 45
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 18782759
- Full Text :
- https://doi.org/10.1074/jbc.M803930200