Cimpean Anisoara, Stefan Cristiana, Gijsbers Rik, Stalmans Willy, Bollen Mathieu
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium.
Biochem J. 2004 Jul 1;381(Pt 1):71-7. doi: 10.1042/BJ20040465.
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.
核苷酸焦磷酸酶/磷酸二酯酶NPP1和NPP2/自分泌运动因子是结构相关的真核外切酶，但具有非常不同的底物特异性。NPP1从各种核苷酸中释放核苷5'-单磷酸，而NPP2主要作为溶血磷脂酶D发挥作用。我们采用结构域交换方法来确定NPP1和NPP2的底物特异性决定因素。与NPP2的N端和C端结构域融合的NPP1催化结构域作为核苷酸磷酸二酯酶具有高活性，但没有显示出任何溶血磷脂酶D活性。相反，NPP2催化结构域与NPP1的N端和/或C端结构域的嵌合体完全无活性。这些数据表明，NPP2的催化结构域以及两端都包含溶血磷脂特异性序列。在NPP1和NPP2的催化结构域内，我们确定了靠近催化位点的残基，这些残基决定了对核苷酸和溶血磷脂的活性。我们还表明，催化位点附近保守的Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly（G/FXGXXG）基序是金属结合所必需的，但不参与底物特异性的决定。我们的数据表明，NPP1和NPP2的不同活性源于整个多肽链中的多种差异。
