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Isolation of a cDNA coding for L-galactono-γ-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants: purification, characterization, cDNA cloning, and expression in yeast

Jens Østergaard UGent, Geert Persiau UGent, Mark W Davey UGent, Guy Bauw UGent and Marc Van Montagu UGent (1997) JOURNAL OF BIOLOGICAL CHEMISTRY. 272(48). p.30009-30016
abstract
L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3; GLDase), an enzyme that catalyzes the final step in the biosynthesis of L-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (Brassica oleracea, var. botrytis) to apparent homogeneity with an overall yield of 1.1%, The purification procedure consisted of anion exchange, hydrophobic interaction, gel filtration, and fast protein liquid chromatography, The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis and showed a pH optimum for activity between pH 8.0 and 8.5, with an apparent K-m of 3.3 mM for L-galactono-gamma-lactone. Based on partial peptide sequence information, polymerase chain reaction fragments were isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated, The deduced mature GLDase contained 509 amino acid residues with a predicted molecular mass of 57,837 Da, Expression of the cDNA in yeast produced a biologically active protein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted with L-galactono-gamma-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of L-ascorbic acid biosynthesis in vivo.
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organization
alternative title
Isolation of a cDNA coding for L-galactono-gamma-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants : purification, characterization, cDNA cloning, and expression in yeast
year
type
journalArticle (original)
publication status
published
subject
keyword
OXIDASE, FLAVIN, TARGETING PEPTIDES, NUCLEOTIDE-SEQUENCE, SACCHAROMYCES-CEREVISIAE, SPINACH LEAVES, ESCHERICHIA-COLI, MITOCHONDRIAL, KEY ENZYME, L-SORBOSONE
journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
J. Biol. Chem.
volume
272
issue
48
pages
30009 - 30016
Web of Science type
Article
ISSN
0021-9258
DOI
10.1074/jbc.272.48.30009
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
185982
handle
http://hdl.handle.net/1854/LU-185982
date created
2004-01-14 13:41:00
date last changed
2016-12-19 15:38:47
@article{185982,
  abstract     = {L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3; GLDase), an enzyme that catalyzes the final step in the biosynthesis of L-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (Brassica oleracea, var. botrytis) to apparent homogeneity with an overall yield of 1.1\%, The purification procedure consisted of anion exchange, hydrophobic interaction, gel filtration, and fast protein liquid chromatography, The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis and showed a pH optimum for activity between pH 8.0 and 8.5, with an apparent K-m of 3.3 mM for L-galactono-gamma-lactone. Based on partial peptide sequence information, polymerase chain reaction fragments were isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated, The deduced mature GLDase contained 509 amino acid residues with a predicted molecular mass of 57,837 Da, Expression of the cDNA in yeast produced a biologically active protein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted with L-galactono-gamma-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of L-ascorbic acid biosynthesis in vivo.},
  author       = {{\O}stergaard, Jens and Persiau, Geert and Davey, Mark W and Bauw, Guy and Van Montagu, Marc},
  issn         = {0021-9258},
  journal      = {JOURNAL OF BIOLOGICAL CHEMISTRY},
  keyword      = {OXIDASE,FLAVIN,TARGETING PEPTIDES,NUCLEOTIDE-SEQUENCE,SACCHAROMYCES-CEREVISIAE,SPINACH LEAVES,ESCHERICHIA-COLI,MITOCHONDRIAL,KEY ENZYME,L-SORBOSONE},
  language     = {eng},
  number       = {48},
  pages        = {30009--30016},
  title        = {Isolation of a cDNA coding for L-galactono-\ensuremath{\gamma}-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants: purification, characterization, cDNA cloning, and expression in yeast},
  url          = {http://dx.doi.org/10.1074/jbc.272.48.30009},
  volume       = {272},
  year         = {1997},
}

Chicago
Østergaard, Jens, Geert Persiau, Mark W Davey, Guy Bauw, and Marc Van Montagu. 1997. “Isolation of a cDNA Coding for L-galactono-γ-lactone Dehydrogenase, an Enzyme Involved in the Biosynthesis of Ascorbic Acid in Plants: Purification, Characterization, cDNA Cloning, and Expression in Yeast.” Journal of Biological Chemistry 272 (48): 30009–30016.
APA
Østergaard, J., Persiau, G., Davey, M. W., Bauw, G., & Van Montagu, M. (1997). Isolation of a cDNA coding for L-galactono-γ-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants: purification, characterization, cDNA cloning, and expression in yeast. JOURNAL OF BIOLOGICAL CHEMISTRY, 272(48), 30009–30016.
Vancouver
1.
Østergaard J, Persiau G, Davey MW, Bauw G, Van Montagu M. Isolation of a cDNA coding for L-galactono-γ-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants: purification, characterization, cDNA cloning, and expression in yeast. JOURNAL OF BIOLOGICAL CHEMISTRY. 1997;272(48):30009–16.
MLA
Østergaard, Jens, Geert Persiau, Mark W Davey, et al. “Isolation of a cDNA Coding for L-galactono-γ-lactone Dehydrogenase, an Enzyme Involved in the Biosynthesis of Ascorbic Acid in Plants: Purification, Characterization, cDNA Cloning, and Expression in Yeast.” JOURNAL OF BIOLOGICAL CHEMISTRY 272.48 (1997): 30009–30016. Print.