Structural and catalytic similarities between nucleotide pyrophosphatases/phosphodiesterases and alkaline phosphatases.
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 metal-coordinating 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.
Gijsbers
R
R
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Ceulemans
H
H
Stalmans
W
W
Bollen
M
M
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D010802:Phylogeny; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; C405782:nucleotide pyrophosphatase - phosphodiesterase I