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Partial purification and identification of GDP-mannose 3", 5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway

Beata Wolucka (UGent) , Geert Persiau (UGent) , Jan Van Doorsselaere (UGent) , Mark W Davey (UGent) , Hans Demol (UGent) , Joël Vandekerckhove (UGent) , Marc Van Montagu (UGent) , Marc Zabeau (UGent) and Wout Boerjan (UGent)
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Abstract
The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, Of L-galactosyl residues, catalyzed by a largely unknown GDP-D-mannose 3 " ,5 " -epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-D-mannose 3 " ,5 " -epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a BLAST search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-D-mannose 5 " -epimerase. The plant GDP-D-mannose 3 " ,5 " -epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.
Keywords
BIOSYNTHESIS, PROTEIN, DEHYDROGENASE, MUR1 GENE, CELL-WALLS, ESCHERICHIA-COLI, L-FUCOSE, L-ASCORBIC-ACID, DE-NOVO SYNTHESIS, MULTIPLE SEQUENCE ALIGNMENT

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MLA
Wolucka, Beata, Geert Persiau, Jan Van Doorsselaere, et al. “Partial Purification and Identification of GDP-mannose 3‘, 5’-epimerase of Arabidopsis Thaliana, a Key Enzyme of the Plant Vitamin C Pathway.” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 98.26 (2001): 14843–14848. Print.
APA
Wolucka, B., Persiau, G., Van Doorsselaere, J., Davey, M. W., Demol, H., Vandekerckhove, J., Van Montagu, M., et al. (2001). Partial purification and identification of GDP-mannose 3“, 5”-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 98(26), 14843–14848.
Chicago author-date
Wolucka, Beata, Geert Persiau, Jan Van Doorsselaere, Mark W Davey, Hans Demol, Joël Vandekerckhove, Marc Van Montagu, Marc Zabeau, and Wout Boerjan. 2001. “Partial Purification and Identification of GDP-mannose 3‘, 5’-epimerase of Arabidopsis Thaliana, a Key Enzyme of the Plant Vitamin C Pathway.” Proceedings of the National Academy of Sciences of the United States of America 98 (26): 14843–14848.
Chicago author-date (all authors)
Wolucka, Beata, Geert Persiau, Jan Van Doorsselaere, Mark W Davey, Hans Demol, Joël Vandekerckhove, Marc Van Montagu, Marc Zabeau, and Wout Boerjan. 2001. “Partial Purification and Identification of GDP-mannose 3‘, 5’-epimerase of Arabidopsis Thaliana, a Key Enzyme of the Plant Vitamin C Pathway.” Proceedings of the National Academy of Sciences of the United States of America 98 (26): 14843–14848.
Vancouver
1.
Wolucka B, Persiau G, Van Doorsselaere J, Davey MW, Demol H, Vandekerckhove J, et al. Partial purification and identification of GDP-mannose 3“, 5”-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 2001;98(26):14843–8.
IEEE
[1]
B. Wolucka et al., “Partial purification and identification of GDP-mannose 3", 5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway,” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 98, no. 26, pp. 14843–14848, 2001.
@article{138840,
  abstract     = {The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, Of L-galactosyl residues, catalyzed by a largely unknown GDP-D-mannose 3 " ,5 " -epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-D-mannose 3 " ,5 " -epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a BLAST search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-D-mannose 5 " -epimerase. The plant GDP-D-mannose 3 " ,5 " -epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.},
  author       = {Wolucka, Beata and Persiau, Geert and Van Doorsselaere, Jan and Davey, Mark W and Demol, Hans and Vandekerckhove, Joël and Van Montagu, Marc and Zabeau, Marc and Boerjan, Wout},
  issn         = {0027-8424},
  journal      = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
  keywords     = {BIOSYNTHESIS,PROTEIN,DEHYDROGENASE,MUR1 GENE,CELL-WALLS,ESCHERICHIA-COLI,L-FUCOSE,L-ASCORBIC-ACID,DE-NOVO SYNTHESIS,MULTIPLE SEQUENCE ALIGNMENT},
  language     = {eng},
  number       = {26},
  pages        = {14843--14848},
  title        = {Partial purification and identification of GDP-mannose 3", 5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway},
  url          = {http://dx.doi.org/10.1073/pnas.011578198},
  volume       = {98},
  year         = {2001},
}

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