Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2.
The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5'-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain.
Cimpean
Anisoara
A
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium.
Stefan
Cristiana
C
Gijsbers
Rik
R
Stalmans
Willy
W
Bollen
Mathieu
M
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D020134:Catalytic Domain / Q000502:physiology; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000737:chemistry / Q000502:physiology; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011755:Pyrophosphatases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D013912:Threonine / Q000502:physiology; D000099189:Lysophospholipase D
D005956:Glucose-6-Phosphate Isomerase; D006023:Glycoproteins; D009097:Multienzyme Complexes; D010455:Peptides; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011993:Recombinant Fusion Proteins; D013912:Threonine; D000099189:Lysophospholipase D; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1