Phosphonoacetate hydrolase from Penicillium oxalicum: purification and properties, phosphate starvation-independent expression, and partial sequencing.
The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed.
Klimek-Ochab
Magdalena
M
Department of Chemistry, 50-370 Wrocław, Poland.
Raucci
Giuseppe
G
Lejczak
Barbara
B
Forlani
Giuseppe
G
D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases; C097254:phosphonoacetate hydrolase