Dataset Information

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.

SUBMITTER: Wolucka BA

PROVIDER: S-EPMC64946 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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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 BA Persiau G G Van Doorsselaere J J Davey M W MW Demol H H Vandekerckhove J J Van Montagu M M Zabeau M M Boerjan W W

Proceedings of the National Academy of Sciences of the United States of America 20011201 26

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 ...[more]

PMID: 11752432

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Project description:D-Enantiomers of proteinogenic amino acids (D-AAs) are found ubiquitously, but the knowledge about their metabolism and functions in plants is scarce. A long forgotten phenomenon in this regard is the D-AA-stimulated ethylene production in plants. As a starting point to investigate this effect, the Arabidopsis accession Landsberg erecta (Ler) got into focus as it was found defective in metabolizing D-AAs. Combining genetics and molecular biology of T-DNA insertion lines and natural variants together with biochemical and physiological approaches, we could identify AtDAT1 as a major D-AA transaminase in Arabidopsis. Atdat1 loss-of-function mutants and Arabidopsis accessions with defective AtDAT1 alleles were unable to produce the metabolites of D-Met, D-Ala, D-Glu, and L-Met. This result corroborates the biochemical characterization, which showed highest activity of AtDAT1 using D-Met as a substrate. Germination of seedlings in light and dark led to enhanced growth inhibition of atdat1 mutants on D-Met. Ethylene measurements revealed an increased D-AA stimulated ethylene production in these mutants. According to initial working models of this phenomenon, D-Met is preferentially malonylated instead of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This decrease of ACC degradation should then lead to the increase of ethylene production. We could observe a reciprocal relation of malonylated methionine and ACC upon D-Met application and significantly more malonyl-methionine in atdat1 mutants. Unexpectedly, the malonyl-ACC levels did not differ between mutants and wild type. With AtDAT1, the first central enzyme of plant D-AA metabolism was characterized biochemically and physiologically. The specific effects of D-Met on ACC metabolism, ethylene production, and plant development of dat1 mutants unraveled the impact of AtDAT1 on these processes; however, they are not in full accordance to previous working models. Instead, our results imply the influence of additional factors or processes on D-AA-stimulated ethylene production, which await to be uncovered.

Dataset Information

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

Publications

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

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