Identification, cloning, and expression of Pseudomonas aeruginosa phosphorylcholine phosphatase gene.
Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.
Massimelli
María J
MJ
Departamento de Biología Molecular, Facultad de Ciencias Exactas, Fisicoquímicas y Naturales, Universidad Nacional de Río Cuarto, 5800, Río Cuarto, Córdoba, Argentina.
Beassoni
Paola R
PR
Forrellad
Marina A
MA
Barra
José L
JL
Garrido
Mónica N
MN
Domenech
Carlos E
CE
Lisa
Angela T
AT
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010767:Phosphorylcholine / Q000378:metabolism*; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity
D004983:Ethanolamines; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010767:Phosphorylcholine; C008644:nitrophenylphosphate; C005448:phosphorylethanolamine; D010744:Phosphoric Monoester Hydrolases