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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.