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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
- Wolucka, B.A., Persiau, G., Doorsselaere, J. van., Davey, M.W., Demol, H., Vandekerckhove, J., Montagu, M. van., Zabeau, M., Boerjan, W.
- Proceedings of the National Academy of Sciences of the United States of America 2001 v.98 no.26 pp. 14843-14848
- Arabidopsis thaliana, biosynthesis, molecular weight, amino acid sequences, isomerases, exons, enzyme activity, introns, ascorbic acid
- 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 BImG 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.