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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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Chicago
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.
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.
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.
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.
@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 {\textacutedbl} ,5 {\textacutedbl} -epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-D-mannose 3 {\textacutedbl} ,5 {\textacutedbl} -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 {\textacutedbl} -epimerase. The plant GDP-D-mannose 3 {\textacutedbl} ,5 {\textacutedbl} -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, Jo{\"e}l 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},
  keyword      = {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{\textacutedbl}, 5{\textacutedbl}-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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