The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence

Abstract

Serine/threonine protein phosphatases have been described in many pathogenic bacteria as essential enzymes involved in phosphorylation-dependent signal transduction pathways and frequently associated with the virulence of these organisms. An inspection of Mycoplasma synoviae genome revealed the presence of a gene (prpC) encoding a putative protein phosphatase of the protein phosphatase 2C (PP2C) subfamily. Here, we report a complete biochemical characterization of M. synoviae phosphatase (PrpC) and the particular role of metal ions in the structure-function relationship of this enzyme. PrpC amino acid sequence analysis revealed that all the residues involved in the dinuclear metal center and the putative third metal ion-coordinating residues, conserved in PP2C phosphatases, are present in PrpC. PrpC is a monomeric protein able to dephosphorylate phospho-substrates with Mn(2+) ions' dependence. Thermal stability analysis demonstrated the enzyme stability at mild temperatures and the influence of Mn(2+) ions in this property. Mass spectrometry analysis suggested that three metal ions bind to PrpC, two of which with an apparent high-affinity constant. Mutational analysis of the putative third metal-coordinating residues, Asp122 and Arg164, revealed that these variants exhibited a weaker binding of manganese ions, and that both mutations affected PrpC phosphatase activity. According to these results, PrpC is a metal-dependent protein phosphatase member with an improved stability in the holo form and with Asp122, possibly implicated in the third metal-binding site, essential to catalytic activity.

CTL0511 from Chlamydia trachomatis Is a Type 2C Protein Phosphatase with Broad Substrate Specificity

“…3). Consistent with other PP2Cs, rCTL0511 exhibits a K m and k cat of 0.95 Ϯ 0.21 mM and 8.4 Ϯ 0.40 s Ϫ1 , respectively (28,34,(37)(38)(39)(40)(41)(42)(43). Other previously reported kinetic values ranged from 0.35 to 5.7 mM pNPP for K m and 0.10 to 7.4 s Ϫ1 for k cat (Table 1).…”

“…In contrast, phosphatase activity was completely abrogated in the presence of sodium pyrophosphate, a general Ser/Thr phosphatase inhibitor. Similar to other bacterial phosphatases, rCTL0511 displayed divalent cation dependent activity that was optimal at alkaline pH levels (34,37,39), a K m of 0.95 Ϯ 0.21 mM pNPP, and a k cat of 8.4 Ϯ 0.40 s…”

“…also assayed in the presence of sodium pyrophosphate, an irreversible inhibitor of Ser/Thr protein phosphatases typically used at a final concentration of 1 mM to 10 mM (34,37). The presence of either okadaic acid or cyclosporine at levels Ͼ200-fold above their respective IC 50 did not inhibit phosphatase activity in comparison to the DMSO control (Fig.…”

“…Both PTPs and BY kinases are noticeably absent in Chlamydia species (65). While several bacterial PP2Cs have been shown to mediate P-Tyr dephosphorylation in vitro, the biological relevance of PP2C-mediated tyrosine dephosphorylation remains unknown, and the need for a tyrosine phosphatase in C. trachomatis is unclear (34,37,66). In Chlamydia, the Hanks'-type kinases PknD and Pkn1 have been functionally validated in C. trachomatis and C. pneumoniae (PknD only) (19,20).…”

Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

“…Similarly, the Kcat values were calculated to be 0.2089 s −1 and 0.093s −1 for REL606 and MG1655 PphC, respectively. Previously reported kinetic values for known bacterial PP2Cs range from 0.35mM to 5.7mM pNPP for Km and 0.1 to 7.4 s −1 for Kcat (22–24, 26, 27, 31, 40–43), indicating that PphC has relatively low phosphatase activity in vitro as compared to previously characterized bacterial PP2C-like phosphatases.…”