The PPP-Family Protein Phosphatases PrpA and PrpB of Salmonella enterica Serovar Typhimurium Possess Distinct Biochemical Properties

Abstract

Salmonella enterica serovar Typhimurium requires Mn2؉ , but only a few Mn 2؉ -dependent enzymes have been identified from bacteria. To characterize Mn 2؉ -dependent enzymes from serovar Typhimurium, two putative PPP-family protein phosphatase genes were cloned from serovar Typhimurium and named prpA and prpB. Their DNA-derived amino acid sequences showed 61% identity to the corresponding Escherichia coli proteins and 41% identity to each other. Each phosphatase was expressed in E. coli and purified to near electrophoretic homogeneity. Both PrpA and PrpB absolutely required a divalent metal for activity. As with other phosphatases of this class, Mn 2؉ had the highest affinity and stimulated the greatest activity. The apparent K a of PrpA for Mn 2؉ of 65 M was comparable to that for other bacterial phosphatases, but PrpB had a much higher affinity for Mn 2؉ (1.3 M). The pH optima were pH 6.5 for PrpA and pH 8 for PrpB, while the optimal temperatures were 45 to 55°C for PrpA and 30 to 37°C for PrpB. Each phosphatase could hydrolyze phosphorylated serine, threonine, or tyrosine residues, but their relative specific activities varied with the specific substrate tested. These differences suggest that each phosphatase is used by serovar Typhimurium under different growth or environmental conditions such as temperature or acidity.MntH, the Salmonella enterica serovar Typhimurium ortholog of the eukaryotic NRAMP proteins (natural resistanceassociated macrophage protein), is a highly selective H ϩ -stimulated Mn 2ϩ transporter (6). Subsequently, the mammalian NRAMP1 protein, expressed in monocytes and macrophages, was shown to mediate marked changes in intracellular Mn 2ϩ in macrophages (5). Transcription of mntH in serovar Typhimurium is induced by hydrogen peroxide and cation starvation, and mutation of mntH makes serovar Typhimurium cells more sensitive to killing by peroxide. These data suggest that Mn 2ϩ is required for maintenance of some physiological functions and that Mn 2ϩ could play a role in virulence, especially in resistance to reactive oxygen species (6). Any role for Mn 2ϩ is presumably mediated by its use as an enzyme cofactor. Surprisingly, very few known enzymes are known to be Mn 2ϩ dependent. In an effort to understand the role of Mn 2ϩ in cell function and pathogenesis, we have begun to characterize each of the few known Mn 2ϩ -cofactored enzymes. One such family is the PPP class of protein phosphatases (2,8,17).PPP phosphatases are metalloenzymes that include the catalytic subunits of mammalian PP1, PP2A, PP2B, and related protein phosphatases. Two divalent metal ions in their active sites catalyze a single-step dephosphorylation reaction. In eukaryotes, PPP phosphatases are involved in regulating multiple cellular functions such as cell division, glycogen metabolism, and muscle contraction. Homologs of PPP phosphatases can be discerned in many bacterial and archaeal genomes (for reviews, see references 8 and 17). All bacterial PPP phosphatases characterized to date require Mn 2ϩ for maximal activat...

Bacterial Acetone Carboxylase Is a Manganese-dependent Metalloenzyme

“…Thus, there are two manganese sites in bacteriophage protein phosphatase: a higher affinity site forming the mononuclear cluster and a lower affinity site that binds an additional manganese to form the dinuclear site (31). It should be noted that this phosphatase will use other metal ions, including nickel(II), in place of manganese and that manganese does not associate with the enzyme throughout purification (51,52). Similarly, arginase is inactive as isolated and is activated by manganese and other metal ions, in the process forming a dinuclear cluster (35).…”

Regulation ofSalmonella entericaSerovar TyphimuriummntHTranscription by H2O2, Fe2+, and Mn2+

“…Second, the MntR gene stop codon overlaps the start codon of the gene encoding putative membrane protein YbiR, b0818 in Blattner E. coli notation (6), whose BLAST hits are annotated variously as carboxylate or arsenate transporters. We are investigating the possibility that YbiR mediates the Mn 2ϩ efflux activity that we observed during our characterization of MntH as an Mn 2ϩ uptake protein (24 (36), and the orthologous protein phosphatases PrpA and PrpB (28,29,39), which modulate repair of envelope damage in E. coli. Metabolic Mn 2ϩ enzymes include ppGpp synthase/hydrolase SpoT, which modulates the bacterial response to amino acid starvation (12,30,37) and phosphoglyceromutase YibO, b3612 in Blattner E. coli notation (6), which is regulated distinctly from the 2,3-bisphosphoglycerate-cofactored GpmA (14,17) and for which a distinctive role is not yet known.…”

Autophosphorylation of the Escherichia coli Protein Kinase Wzc Regulates Tyrosine Phosphorylation of Ugd, a UDP-glucose Dehydrogenase

“…The question therefore arises whether E. coli possesses additional PTPs able to efficiently demodify phosphorylated Ugd and/or Wzc phosphorylated at Tyr 569 . The E. coli proteins PrpA and PrpB (37,38), which belong to the PPP family of protein phosphatases, could be such candidates. Although members of this family were first described as phosphoserine/phosphothreonine protein phosphatases, some of them, including PrpA and PrpB, have been found to also dephosphorylate phosphotyrosine (39).…”