title: 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 degrees C, respectively. The Km value for PnPy was 19 +/- 3.5 microM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 microM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the Km values were 3.5 +/- 1.4 microM for Mg2+, 16 +/- 5 nM for Mn2+, 3.0 +/- 1.5 microM for Zn2+, and 1.2 +/- 0.2 microM for Co2+.
authors.affiliation: Institute of Applied Microbiology, University of Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nakashita
authors.forename: A
authors.initials: A
authors.lastname: Shimazu
authors.forename: T
authors.initials: T
authors.lastname: Hidaka
authors.forename: H
authors.initials: H
authors.lastname: Seto
mesh_terms: D002244:Carbon / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000187:drug effects / Q000302:isolation & purification*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010758:Phosphorus / Q000737:chemistry; D017875:Phosphotransferases (Phosphomutases); D011549:Pseudomonas / Q000201:enzymology*
chemical_list: D010758:Phosphorus; D002244:Carbon; D007535:Isomerases; D017875:Phosphotransferases (Phosphomutases); C057167:phosphoenolpyruvate mutase; D008274:Magnesium