Publications

• The purification and characterization of phosphonopyruvate hydrolase, a novel carbon-phosphorus bond cleavage enzyme from Variovorax sp Pal2.

  Kulakova A.N., Wisdom G.B., Kulakov L.A., Quinn J.P.

  Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine o ... >> More

  Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7-7.0. The enzyme had a Km of 0.53 mm for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond. << Less

  J. Biol. Chem. 278:23426-23431(2003) [PubMed] [EuropePMC]

• Initial in vitro characterisation of phosphonopyruvate hydrolase, a novel phosphate starvation-independent, carbon-phosphorus bond cleavage enzyme in Burkholderia cepacia Pal6.

  Ternan N.G., Hamilton J.T., Quinn J.P.

  A novel, inducible carbon-phosphorus bond cleavage enzyme, phosphonopyruvate hydrolase, was detected in cell-free extracts of Burkholderia cepacia Pal6, an environmental isolate capable of mineralising L-phosphonoalanine as carbon, nitrogen and phosphorus source. The activity was induced only in t ... >> More

  A novel, inducible carbon-phosphorus bond cleavage enzyme, phosphonopyruvate hydrolase, was detected in cell-free extracts of Burkholderia cepacia Pal6, an environmental isolate capable of mineralising L-phosphonoalanine as carbon, nitrogen and phosphorus source. The activity was induced only in the presence of phosphonoalanine, did not require phosphate starvation for induction and was uniquely specific for phosphonopyruvate, producing equimolar quantities of pyruvate and inorganic phosphate. The native enzyme had a molecular mass of some 232 kDa and showed activation by metal ions in the order Co2+ > Ni2+ > Mg2+ > Zn2+ > Fe2+ > Cu2+. Temperature and pH optima in crude cell extracts were 50 degrees C and 7.5, respectively, and activity was inhibited by EDTA, phosphite, sulfite, mercaptoethanol and sodium azide. Phosphonopyruvate hydrolase is the third bacterial C-P bond cleavage enzyme reported to date that proceeds via a hydrolytic mechanism. << Less

  Arch Microbiol 173:35-41(2000) [PubMed] [EuropePMC]

• Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: new insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily.

  Chen C.C., Han Y., Niu W., Kulakova A.N., Howard A., Quinn J.P., Dunaway-Mariano D., Herzberg O.

  Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia ... >> More

  Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range. The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg2+ binding first (Kd = 140 +/- 40 microM), are kcat = 105 +/-2 s(-1) and P-pyr Km = 5 +/-1 microM. PEP (slow substrate kcat = 2 x 10(-4) s(-1)), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 +/-0.1 mM, 17 +/- 1 microM, and 210 +/-10 microM, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (alpha/beta)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue. << Less

  Biochemistry 45:11491-11504(2006) [PubMed] [EuropePMC]