This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakashita, H Articles by Seto, H Search for Related Content PubMed PubMed Citation Articles by Nakashita, H Articles by Seto, H

research-article

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

Institute of Applied Microbiology, University of Tokyo, Japan.

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+.

This article has been cited by other articles:

• Kulakova, A. N., Wisdom, G. B., Kulakov, L. A., Quinn, J. P. (2003). The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2. J. Biol. Chem. 278: 23426-23431 [Abstract] [Full Text]  
• Obojska, A., Ternan, N. G., Lejczak, B., Kafarski, P., McMullan, G. (2002). Organophosphonate Utilization by the Thermophile Geobacillus caldoxylosilyticus T20. Appl. Environ. Microbiol. 68: 2081-2084 [Abstract] [Full Text]  
• Ternan, N. G., McGrath, J. W., Quinn, J. P. (1998). Phosphoenolpyruvate Phosphomutase Activity in an L-Phosphonoalanine-Mineralizing Strain of Burkholderia cepacia. Appl. Environ. Microbiol. 64: 2291-2294 [Abstract] [Full Text]  
• Kim, A., Kim, J., Martin, B. M., Dunaway-Mariano, D. (1998). Isolation and Characterization of the Carbon-Phosphorus Bond-forming Enzyme Phosphoenolpyruvate Mutase from the Mollusk Mytilus edulis. J. Biol. Chem. 273: 4443-4448 [Abstract] [Full Text]