A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis.
Ascorbate is a critical compound in plants and animals. Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants. However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist. We describe a specific L-galactose-1-phosphate (L-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries. The enzyme had a native molecular mass of approximately 65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze L-gal-1-P. The activity had a pH optimum of 7.0, a K(-M(L-gal-1-P) of 20-40 microM and a Ka(Mg2+) of 0.2 mM. The activity was inhibited by Mg2+ at concentrations >2 mM. The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme. The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography-MS. One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase. We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P. The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(L-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme. The data are discussed in terms of the pathway to ascorbate biosynthesis in plants.
Laing
William A
WA
Gene Technologies Sector, Horticultural and Food Research Institute of New Zealand, Ltd., PB 92169, Auckland, New Zealand. wlaing@hortresearch.co.nz
Bulley
Sean
S
Wright
Michele
M
Cooney
Janine
J
Jensen
Dwayne
D
Barraclough
Di
D
MacRae
Elspeth
E
D029042:Actinidia / Q000201:enzymology* / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics; D001205:Ascorbic Acid / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D010744:Phosphoric Monoester Hydrolases; D001205:Ascorbic Acid