Arabidopsis thaliana PECP1: enzymatic characterization and structural organization of the first plant phosphoethanolamine/phosphocholine phosphatase.
Maintenance of cellular phosphate homeostasis is crucial for primary and energy metabolism. In plants, low exogenous phosphate availability activates adaptive responses that include the immediate liberation of Pi from phosphorylated metabolites by yet uncharacterized intracellular phosphatases. Based on transcriptional analyses, the Arabidopsis thaliana gene At1g17710, a member of the HAD (Haloacid Dehalogenase) superfamily, was one of the most promising candidates. Here, we show by recombinant protein production and analysis of purified protein that the gene At1g17710 encodes a phosphoethanolamine/phosphocholine phosphatase (EC 3.1.3.75). Thus, the gene product was termed AtPECP1. The present study demonstrates that the Mg(2+)-dependent enzyme exhibits pronounced specificity for both substrates. The enzyme displays a broad pH optimum ranging from pH 6 to pH 8. Comparison of K(m) values indicates a slightly higher affinity for phosphocholine (0.44 mM) than for phosphoethanolamine (1.16 mM). The catalytic efficiency, however, is markedly higher for phosphoethanolamine than for phosphocholine being 1.06 × 10(4)M(-1)s(-1) and 2.34 × 10(3)M(-1)s(-1), respectively. Size exclusion chromatography, native gel electrophoresis and SAXS experiments with recombinant protein clearly point to a rapid monomer-dimer equilibrium of protein subunits. Given its established substrate specificity the enzyme is likely to be involved in the liberation of inorganic phosphate from intracellular sources and is especially in demand under phosphate-deprived conditions.
Biocenter, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Anett
A
May
Michael
M
Spinka
Margret
M
Köck
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D004983:Ethanolamines / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics; D010767:Phosphorylcholine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D004983:Ethanolamines; D010710:Phosphates; D011994:Recombinant Proteins; D010767:Phosphorylcholine; C005448:phosphorylethanolamine; C571576:PECP1 protein, Arabidopsis; C054085:phosphocholine phosphatase; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium