Purification and characterization of phosphoenolpyruvate phosphomutase from Pseudomonas gladioli B-1

Abstract

Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B-1 isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by cleaving the C-P bond of PnPy, but the C-P bond-forming reaction is physiologically significant. The C-P bond-forming activity of PEPPM was confirmed with a purified protein. The molecular mass of the native enzyme was estimated to be 263 and 220 kDa by gel filtration and polyacrylamide gel electrophoresis, respectively. A subunit molecular mass of 61 kDa was determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, indicating that the native protein was a tetramer. The optimum pH and temperature were 7.5 to 8.0 and 40°C, respectively. The K(m) value for PnPy was 19 ± 3.5 μM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 μM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the K(m) values were 3.5 ± 1.4 μM for Mg2+, 16 ± 5 nM for Mn2+, 3.0 ± 1.5 μM for Zn2+, and 1.2 ± 0.2 μM for Co2+.