title: Partial purification and identification of GDP-mannose 3",5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway.
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.
authors.affiliation: 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
authors.forename: B A
authors.initials: BA
authors.lastname: Wolucka
authors.forename: G
authors.initials: G
authors.lastname: Persiau
authors.forename: J
authors.initials: J
authors.lastname: Van Doorsselaere
authors.forename: M W
authors.initials: MW
authors.lastname: Davey
authors.forename: H
authors.initials: H
authors.lastname: Demol
authors.forename: J
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: M
authors.initials: M
authors.lastname: Van Montagu
authors.forename: M
authors.initials: M
authors.lastname: Zabeau
authors.forename: W
authors.initials: W
authors.lastname: Boerjan
mesh_terms: 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
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C019192:GDPmannose 3,5-epimerase; D001205:Ascorbic Acid