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

Abstract

Phosphoenolpyruvate phosphomutv-'^(PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to pr fhonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by deaving 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. value for PnPy was 19 ± 3.5 ,M, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 ,uM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the K. values were 3.5 ± 1.4 FM for Mge+, 16 + 5 nM for Mn+, 3.0 ± 1.5 PM for Zn2+, and 1.2 ± 0.2 P1M for Co2+.Since the discovery of the first natural C-P compound, 2-aminoethylphosphonic acid (10), various metabolites with C-P bonds have been isolated and the presence of phosphonates and phosphinates in a variety of biological systems has been established. Extensive studies on their unique C-P bond formation mechanisms (1,8, 9,20,23,24) indicated that the first step of the C-P bond formation pathway is the intramolecular rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (PnPy) and that the equilibrium of this reaction heavily favors the formation of the phosphate ester ( Fig.

Isolation and Characterization of the Carbon–Phosphorus Bond-forming Enzyme Phosphoenolpyruvate Mutase from the MolluskMytilus edulis

“…1). The fosfomycin pathway PEP mutase-encoding gene from S. wedmorensis (14) has been sequenced, and the enzyme has been purified/ characterized from a probable fosfomycin-producing strain, Pseudomonas gladioli (25). The bialaphos pathway PEP mutase-encoding gene from S. hygroscopicus (24) has been sequenced, but because of its instability, the S. hygroscopicus enzyme has only been partially purified (19).…”

Organophosphonate Utilization by the Thermophile Geobacillus caldoxylosilyticus T20

“…Additionally, the COP bond of phosphonopyruvate can be intramolecularly rearranged to form a phosphate ester, phosphoenolpyruvate, by the action of the enzyme phosphoenolpyruvate phosphomutase (17). However, COP bond cleavage is not the only route by which organophosphonate biodegradation may proceed with both transaminases (22) and oxidoreductases (4), acting on parts of organophosphonate molecules other than the COP bond; indeed, microorganisms that degrade organophosphonates without COP bond cleavage have been described in recent years (15,23).…”

The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2

“…The ability of the enzyme to catalyze the intramolecular rearrangement of phosphonopyruvate to phosphoenolpyruvate was examined using the coupled ADP-pyruvate kinase-NADHlactate dehydrogenase system of Nakashita et al (17); this revealed a rate of phosphoenolpyruvate formation some 1000-fold lower than that of pyruvate and P i . Phosphoenolpyruvate did not itself serve as a substrate for phosphonopyruvate hydrolase.…”