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.
Wolucka
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Departments of Molecular and Plant Genetics and Biochemistry, Faculty of Medicine, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. bewol@gengenp.rug.ac.be
Persiau
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Van Doorsselaere
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Davey
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Demol
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Vandekerckhove
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Van Montagu
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Zabeau
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Boerjan
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D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C019192:GDPmannose 3,5-epimerase; D001205:Ascorbic Acid