Structural and Catalytic Similarities between Nucleotide Pyrophosphatases/Phosphodiesterases and Alkaline Phosphatases

Abstract

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) generate nucleoside 5-monophosphates from a variety of nucleotides and their derivatives. Here we show by data base analysis that these enzymes are conserved from eubacteria to higher eukaryotes. We also provide evidence for the existence of two additional members of the mammalian family of ecto-NPPs. Homology searches and alignment-assisted mutagenesis revealed that the catalytic core of NPPs assumes a fold similar to that of a superfamily of phospho-/sulfo-coordinating metalloenzymes comprising alkaline phosphatases, phosphoglycerate mutases, and arysulfatases. Mutation of mouse NPP1 in some of its predicted metalcoordinating residues (D358N or H362Q) or in the catalytic site threonine (T238S) resulted in an enzyme that could still form the nucleotidylated catalytic intermediate but was hampered in the second step of catalysis. We also obtained data indicating that the ability of some mammalian NPPs to auto(de)phosphorylate is due to an intrinsic phosphatase activity, whereby the enzyme phosphorylated on Thr-238 represents the covalent intermediate of the phosphatase reaction. The results of site-directed mutagenesis suggested that the nucleotide pyrophosphatase/phosphodiesterase and the phosphatase activities of NPPs are mediated by a single catalytic site.

Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity

“…Interestingly, we found in the present study that its activity against PAF is increased by Zn 2+ at 0.25 mM and inhibited by EDTA. The Zn 2+ dependency resembles that of other NPP members [26,31]. However, the activity of alk-SMase against PAF could also be inhibited by Zn 2+ at concentrations higher than 0.25 mM and by mutations of the metal-binding sites, similar to its activity against SM.…”

“…A general property of NPPs is the dependency of the activity on metal ions, particularly Zn 2+ . There are six amino acid residues forming two predicted metal-binding sites in this enzyme family, which were conserved also in alk-SMase [17,26]. An interesting finding that reveals a unique property of alk-SMase is that the activity against SM is not dependent on metal ions, but is inhibited by Zn 2+ and also by mutations within the metal-binding site.…”

“…Ca 2+ had little effect on the activity against PAF and Mg 2+ showed a mild inhibitory effect. Asp 199 and Asp 246 are two amino acid residues that participate in the formation of the two putative metal ion-binding sites in alk-SMase and NPP [17,26]. These sites are important for activity against PAF in the presence of 0.25 mM Zn 2+ , as shown in the bottom panel of Figure 8.…”

“…Alk-SMase belongs to the NPP family and a common property of most NPP members is the dependency of the activity on metal ions, particularly Zn 2+ [26]. However, the activity of alk-SMase against SM is not dependent on metal ions and is inhibited by Zn 2+ and by mutations of the metal-binding sites under the optimal conditions [24].…”

Extracellular nucleotide metabolism and signaling in the pathophysiology of articular cartilage

“…NPP3 (PD-1␤, B-10, gp130 ) was immunodetected on the apical membrane of rat hepatocytes (45). NPP4 and NPP5 are currently described as putative E-NPPs (46). Although defined as intrinsic membrane proteins, soluble forms of NPP1, NPP2␣, and NPP3 are detected in plasma (41,47).…”