PrpZ, a Salmonella enterica serovar Typhi serine/threonine protein phosphatase 2C with dual substrate specificity.
Genes encoding eukaryotic-type protein kinases and phosphatases are present in many bacterial genomes. An ORF encoding a polypeptide with homology to protein phosphatases 2C (PP2Cs) was identified in the genomes of Salmonella enterica serovar Typhi strains CT18 and Ty2. This protein, termed PrpZ, is the first PP2C to be identified in enterobacteria. Analysis of the amino acid sequence revealed two distinct domains: the N-terminal segment containing motifs of the catalytic domain of PP2Cs and the C-terminal segment with unknown function. PrpZ was expressed in Escherichia coli as a histidine-tagged fusion protein (PrpZ(His)) and the purified protein was analysed for its ability to dephosphorylate various substrates. Using p-nitrophenyl phosphate as a substrate, optimal PrpZ(His) activity was observed at pH 9.5, with a strong preference for Mn(2+) over Mg(2+). Activity of PrpZ(His) was inhibited by EDTA, sodium fluoride, sodium phosphate and sodium pyrophosphate but unaffected by okadaic acid, indicating that PrpZ is a PP2C. Using synthetic phosphopeptides as substrates, PrpZ(His) could hydrolyse phosphorylated serine, threonine or tyrosine residues, with the highest catalytic efficiency (k(cat)/K(m)) for the threonine phosphopeptide. With phosphorylated myelin basic protein (MBP) as the substrate, Mn(2+) was only twofold more efficient than Mg(2+) in stimulating PrpZ(His) activity at pH 8.0. The ability of PrpZ(His) to remove the phosphoryl group from phosphotyrosine residues was confirmed by measuring the release of inorganic phosphate from phospho-Tyr MBP. Together, these data indicate that PrpZ has all the features of a PP2C with dual substrate specificity in vitro.
Lai
Sio Mei
SM
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montréal, Québec, Canada H3A 2B4.
Moual
Hervé Le
HL
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montréal, Québec, Canada H3A 2B4.
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D016366:Open Reading Frames; D009943:Organophosphorus Compounds / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071636:Protein Phosphatase 2C; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012485:Salmonella typhi / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D004269:DNA, Bacterial; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; C008644:nitrophenylphosphate; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C