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PrpZ, a Salmonella enterica serovar Typhi serine/threonine protein phosphatase 2C with dual substrate specificity

Department of Microbiology and Immunology, McGill University, 3775 University Street, Montréal, Québec, Canada H3A 2B4

Correspondence
Hervé Le Moual
herve.le-moual{at}mcgill.ca

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 Mn²⁺ over Mg²⁺. 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, Mn²⁺ was only twofold more efficient than Mg²⁺ 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.