Characterization of an acid phosphatase responsible for hydrolysis of pyridoxal 5'-phosphate in tobacco plants.
Pyridoxal 5'-phosphate (PLP), the active form of vitamin B(6), is an important cofactor for many enzymatic reactions. PLP is also a very reactive molecule, and the hydrolysis of PLP is crucial for controlling intracellular PLP concentrations. However, little is known about the enzymatic hydrolysis of PLP in plants. In this study, a novel acid phosphatase was purified from tobacco leaves and characterized by using PLP as a substrate. This phosphatase was purified 180-fold with a yield of 28% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose FF, Sephadex G-100 and SP Sephadex C-25. Our data revealed that the purified enzyme was a dimer with a molecular mass of approximately 50 kDa. The purified phosphatase had maximum catalytic activity at pH 5.5, and displayed optimal activity at 50 °C. The enzyme required divalent metal ion for activity, and Mg(2+), among a few tested cations, was the most effective for catalysis under saturating substrate concentrations. The activity of the purified phosphatase was inhibited by molybdate, fluoride and EDTA, but was not inhibited by levamisole and tartrate. The phosphatase hydrolyzed a broad range of substrates at different rates, and the hydrolysis of PLP was competitively inhibited by ATP, pNPP, and by the reaction products, PL and inorganic phosphate. The phosphatase had a Km of 0.24 mM and a Vmax of 2.76 μmol/min/mg with PLP. When pyridoxamine 5'-phosphate or pyridoxine 5'-phosphate was tested as a substrate, the phosphatase activity was reduced by 50%. Our study suggests that the enzyme is a nonspecific acid phosphatase responsible for hydrolysis of all three phosphorylated B(6) vitamers in tobacco plants.
Key Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of China.
ShuoHao
S
Huang
JianYun
J
Zhang
YaPing
Y
Ma
Shu
S
Wei
LongQuan
L
Huang
D000135:Acid Phosphatase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D010940:Plant Proteins / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D013696:Temperature; D014026:Nicotiana / Q000201:enzymology* / Q000378:metabolism*
D010940:Plant Proteins; D011732:Pyridoxal Phosphate; D000135:Acid Phosphatase; C024593:pyridoxine 5-phosphate