Molecular Cloning and Characterization of L-Galactose-1-phosphate Phosphatase from Tobacco (Nicotiana tabacum)

• Published in: Bioscience, biotechnology, and biochemistry Vol. 76; no. 6; pp. 1155 - 1162
• Main Authors: SAKAMOTO, Shingo, FUJIKAWA, Yukichi, TANAKA, Nobukazu, ESAKA, Muneharu
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 2012; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Allium cepa; Amino Acid Motifs; ascorbate biosynthesis; Ascorbic Acid - biosynthesis; Binding Sites; Biological and medical sciences; Calcium - metabolism; Fundamental and applied biological sciences. Psychology; Galactosephosphates - metabolism; gene expression; Green Fluorescent Proteins; Hydrogen-Ion Concentration; Inositol Phosphates - metabolism; L-galactose-1-phosphate phosphatase; Lithium - metabolism; Magnesium - metabolism; Molecular Sequence Data; Nicotiana - enzymology; Nicotiana - genetics; Nicotiana tabacum; Onions - genetics; Onions - metabolism; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - metabolism; Phylogeny; Plant Proteins - genetics; Plant Proteins - metabolism; Protoplasts - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Substrate Specificity; tobacco BY-2 cell; Phosphates; L-galactose-1-phosphate phosphatase; tobacco BY-2 cell; Enzyme; Phosphoric monoester hydrolases; Esterases; Biosynthesis; Gene expression; Nicotiana tabacum; ascorbate biosynthesis; Characterization; Dicotyledones; Angiospermae; Hydrolases; Spermatophyta; Solanaceae; Molecular cloning; Galactose
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.110995

L-Galactose-1-phosphate phosphatase (GPPase) is an enzyme involved in ascorbate biosynthesis in higher plants. We isolated a cDNA encoding GPPase from tobacco, and named it NtGPPase. The putative amino acid sequence of NtGPPase contained inositol monophosphatase motifs and metal binding sites. Recombinant NtGPPase hydrolyzed not only L-galactose-1-phosphate, but also myo-inositol-1-phosphate. The optimum pH for the GPPase activity of NtGPPase was 7.5. Its enzyme activity required Mg 2+ , and was inhibited by Li + and Ca 2+ . Its fluorescence, fused with green fluorescence protein in onion cells and protoplasts of tobacco BY-2 cells, was observed in both the cytosol and nucleus. The expression of NtGPPase mRNA and protein was clearly correlated with L-ascorbic acid (AsA) contents of BY-2 cells during culture. The AsA contents of NtGPPase over expression lines were higher than those of empty lines at 13 d after subculture. This suggests that NtGPPase contributes slightly to AsA biosynthesis.