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

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.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.A