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

Abstract

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 Co 2؉ -dependent and showed a pH optimum of 6.7-7.0. The enzyme had a K m 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.

Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

“…Alternatively, phosphonoacetaldehyde is oxidized to phosphonoacetate catalyzed by phosphonoacetaldehyde dehydrogenase (59). Also, hydrolytic activities for phosphonoacetate (59,(86)(87)(88)(89)(90) as well as for phosphonopyruvate (91) have been described, and a hydrolytic activity for phosphonoformaldehyde has been suggested (21). Phosphonopyruvate in turn may be formed by the activities of phosphoenolpyruvate phosphomutase or phosphoalanine:2-oxoglutarate transaminase (91)(92)(93).…”

Phosphonate metabolism in Helicobacter pylori

“…The presence of Mg 2? increases catabolism in both the phosphonatase and the phosphonopyruvate hydrolase pathways (Kononova and Nesmeyanova 2002;Kulakova et al 2003;Ternan et al 2000). The effects of Mg 2?…”

“…, and Mg 2? whilst activity decreased with EDTA Kulakova et al 2003). The fourth known pathway for phosphonate metabolism is the C-P lyase pathway.…”

“…The second enzyme, phophonopyruvate hydrolase, was purified from Burkholderia cepacia and from Variovorax spp. (Kulakova et al 2003) with molecular masses of 232 and 63 kDa, respectively. In both cases metabolic activity increased with the addition of Co 2?…”

Phosphate-independent utilization of phosphonoacetic acid as sole phosphorus source by a psychrophilic strain of Geomyces pannorum P15

“…In most cases, phosphonate utilization by bacteria was found to depend upon the phosphate status of the cell with several species where the enzymes expression seem independent of the availability of other P source (Ford et al 2010; Kulakova et al 2003; McMullan and Quinn 1994). …”