The purification and characterization of phosphonopyruvate hydrolase, a novel carbon-phosphorus bond cleavage enzyme from Variovorax sp Pal2.
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.
Kulakova
Anna N
AN
School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland.
Wisdom
G Brian
GB
Kulakov
Leonid A
LA
Quinn
John P
JP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020562:Betaproteobacteria / Q000201:enzymology*; D002845:Chromatography; D003035:Cobalt / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019143:Evolution, Molecular; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D007522:Isocitrate Lyase; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D003035:Cobalt; D006867:Hydrolases; C476343:phosphonopyruvate hydrolase; D007522:Isocitrate Lyase