Molecular cloning and characterization of L-galactose-1-phosphate phosphatase from tobacco (Nicotiana tabacum).
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 Mg2+, and was inhibited by Li+ and Ca2+. 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.
Graduate School of Biosphere Sciences, Hiroshima University, Hiroshima, Japan.
Shingo
S
Sakamoto
Yukichi
Y
Fujikawa
Nobukazu
N
Tanaka
Muneharu
M
Esaka
D020816:Amino Acid Motifs; D001205:Ascorbic Acid / Q000096:biosynthesis; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D005695:Galactosephosphates / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism*; D008094:Lithium / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D019697:Onions / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D005695:Galactosephosphates; D007295:Inositol Phosphates; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C002647:inositol 1-phosphate; C029973:galactose-1-phosphate; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; D001205:Ascorbic Acid; D002118:Calcium