The promiscuous ectonucleotidase NPP1: molecular insights into substrate binding and hydrolysis

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 3; pp. 603 - 614
• Main Authors: Namasivayam, Vigneshwaran, Lee, Sang-Yong, Müller, Christa E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2017
• Subjects: 2′,3″-cGAMP; adenosine monophosphate; adenosine triphosphate; Adenosine Triphosphate - metabolism; Amino Acids - metabolism; Binding Sites - physiology; cAMP; Cancer immunotherapy; cyclic AMP; Cyclic AMP - metabolism; Cyclic GMP - metabolism; Dinucleoside Phosphates - metabolism; drugs; enzymes; guanosine monophosphate; Humans; Hydrolysis; mice; mutagenesis; nucleosides; Nucleotide pyrophosphatase 1; Phosphoric Diester Hydrolases - metabolism; Protein Binding - physiology; Pyrophosphatases - metabolism; Substrate promiscuity; Substrate Specificity; Thymidine Monophosphate - metabolism; uridine triphosphate; Uridine Triphosphate - metabolism; X-radiation; AP4A; CE; CHES; cAMP; Cancer immunotherapy; p-Nph-5′-TMP; 3′,3″-cGAMP; NPP; Nucleotide pyrophosphatase 1; 2′,3″-cGAMP; 3′,3″-cdiGMP; Substrate promiscuity; STING; 3′,3″-cdiIMP
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.12.019

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) represents the main subtype of the NPP family of nucleotide hydrolyzing enzymes. The ecto-enzyme hydrolyzes structurally diverse substrates and has recently been proposed as a drug target for immuno-oncology. To get more insights into the nature of the promiscuity of NPP1, we investigated its substrate preferences employing a broad range of natural nucleotides including ATP, UTP, diadenosine tetraphosphate (AP4A), cAMP, and cyclic guanosine-(2′,5′)-monophosphate-adenosine-(3″,5″)-monophosphate (2′,3″-cGAMP), as well as the artificial substrate p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP). Despite their diverse structures, all substrates were converted to nucleoside 5′-monophosphates; 2′,3″-cGAMP yielded exclusively the nucleoside 5′-monophosphates AMP and GMP. In contrast, 3′,3″-bridged cyclic dinucleotides were not hydrolyzed. ATP was the most efficiently hydrolyzed substrate of NPP1, followed by AP4A and 2′,3″-cGAMP. UTP, cAMP and p-Nph-5′-TMP were much poorer substrates. A homology model of the human NPP1 was built based on the X-ray structure of its mouse orthologue. Docking studies were performed based on previously published mutagenesis data to rationalize the interactions of the different substrates and to explain the enzyme's preferences. The results provide an improved understanding of the interactions of NPP1 with its diverse substrates and will contribute to the validation of NPP1 as a drug target. [Display omitted] •Substrate preferences of NPP1 were analyzed by combining biochemical experiments, homology modeling and docking studies.•All nucleotides are hydrolyzed to nucleoside 5’-monophosphates.•The mechanism of 2’,3’’-cGAMP hydrolyis by NPP1 was elucidated.•The cyclic dinucleotide 2’,3’’-cGAMP yields 5’-AMP and 5’-GMP, while 3’,3’’-bridged cyclic dinucleotides are not hydrolyzed.