Structure and Mutational Analysis of the PhoN Protein of Salmonella typhimurium Provide Insight into Mechanistic Details

Abstract

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.

Polar Localization of PhoN2, a Periplasmic Virulence-Associated Factor of Shigella flexneri, Is Required for Proper IcsA Exposition at the Old Bacterial Pole

“…Among the relevant characteristics of this group of enzymes there is the presence of a N-terminal PPPP motif which was found to be highly conserved among all A-NSAPs analyzed so far. Crystallographic data of the A-NSAP PhoN from Salmonella enterica serovar Typhimurium indicated that the invariant residue Y154 stabilizes the loop harboring the active site residues by binding to the distal P40 residue of the PPPP motif [24], [26], [45].…”

“…Moreover, crystallographic analysis of the PhoN, an A-NSAP from Salmonella typhimurium , and of EB-NSAP, an A-NSAP from Escherichia blattae , indicated that amino acid substitutions within the PPPP motif highly influence the conformational stability (and thus the catalytic activity) of these two proteins. The loss of the conformational stability was shown to be likely due to the loss of an hydrogen bond between the PPPP stretch and an invariant Y residue (Y155 in PhoN2) [26], [45]. These findings prompted us to investigate the role of the Y155 residue of PhoN2-HA in protein stability.…”

“…The mechanism by which PhoN2 influences ABM and plaque size has not been elucidated yet. However, since PhoN2 possesses an exposed N-terminal polyproline ( 43 PPPP 46 ) sequence and proline-rich motifs have been reported to be involved in inter- and intra-molecular interactions, in protein folding and essential for virulence of various intracellular pathogens [22], [23], [24], [25], [26], we hypothesized that the 43 PPPP 46 motif of PhoN2 could directly interact with IcsA or indirectly with unknown accessory proteins necessary in assisting proper polar IcsA exposition [21].…”

Proposed Carrier Lipid-binding Site of Undecaprenyl Pyrophosphate Phosphatase from Escherichia coli

“…This family also contains PhoN protein, vanadium-dependent chloroperoxidases, glucose-6-phosphatase, and lipid phosphate phosphatases (50 -53). The reaction mechanism has been well defined in the PhoN protein by x-ray structural and mutagenesis studies (50). It is generally believed that the histidine initiates a nucleophilic attack at the phosphorus center to form a phosphohistidine intermediate, and the second histidine acting as a general acid/ base facilitates the release of the phosphate.…”

Continuous‐Flow Reactor‐Based Enzymatic Synthesis of Phosphorylated Compounds on a Large Scale

“…Moreover, it allows for continuous processes and easy separation from the products, and thus opens the way to industrial large‐scale applications 15. In fact, in the last decade continuous reactors have progressively substituted batch syntheses, since they help to save time and money in the most challenging “post‐reaction” operations and allow automation, multistep syntheses, and scaling‐up 16…”