The nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes are classified as pyrophosphatases or phosphodiesterases depending on the type of substrates they hydrolyze. NPP1-3 are pyrophosphatases that cleave inorganic phosphates from nucleotides and their derivatives [1
]. In contrast, NPP2, NPP6 and NPP7 are phosphodiesterases that hydrolyze phosphodiester bonds in lipids and their derivatives [2
]. NPP4 and NPP5 are yet to be characterized in terms of substrates and types of activity. NPP2 is the only one of the seven mammalian NPP family members that is known to possess both pyrophosphatase and phosphodiesterase activities [4
]. NPP2 is also the only mammalian family member to have been characterized by X-ray crystallography [5
]. As a phosphodiesterase it exhibits a lysophospholipase D (LPLD) activity, in contrast to NPP6 and NPP7 which exhibit lysophospholipase C (LPLC) activity. Besides these specific activities, NPP family members also exhibit distinct substrate preference profiles but no clear structural basis for the preferences has been proposed [1
]. NPP family members are of considerable interest due to their role in biological functions ranging from bone mineralization to cancer [4
]. NPP7 specifically has been linked with anti-inflammatory and anti-tumorigenic activities through its influence on the conversion of SM to ceramide [9
], however homozygous knockout of NPP7 in mice did not lead to spontaneous tumorigenesis although lipid digestion and absorption was altered [12
]. Mutagenic deletion of a segment from NPP7 including H353, a residue involved in chelating one of the essential divalent metal cations, has been identified in the rapidly proliferating colon cancer HT-29 cell line [13
NPP7 hydrolyzes lysophosphatidylcholine (LPC, Figure ), platelet activating factor (PAF, Figure ) and sphingomyelin (SM, Figure ) with a lysophospholipase C activity but exhibits a preference for SM over LPC and PAF [10
]. Several studies have examined the roles of specific amino acids in the hydrolysis of SM. Three of the conserved metal-chelating residues from the NPP family (D199 and D246 [10
], as well as H353 [13
]) have been mutated to alanine in NPP7, and each mutant was devoid of sphingomyelinase activity. The conserved catalytic threonine residue, T75, has also been mutated to alanine with the expected loss of sphingomyelinase activity [14
]. Additional amino acids near the catalytic residue have been mutated in attempts to change substrate specificity to include nucleotide pyrophosphates, including M74K, S76F, and C78N [14
]. These mutations all eliminated sphingomyelinase activity without producing pyrophosphatase activity. It is unclear whether the mutated proteins were properly folded, although expression levels were comparable to wild type. More recently, mutations based on a comparative model of NPP7 were performed [15
]. In contrast to the result obtained upon mutation of M74 to K, mutation of M74 to L enhanced sphingomyelinase activity, again without gain of pyrophosphatase activity. The model displayed close interaction between F275 and the choline methyl groups, best described as a cation-π interaction. Mutation of F275 to glycine nearly eliminated sphingomyelinase activity. The model displayed no close interaction between F141 and substrates, however, the F141S mutation showed activity nearly as poor as the F275G mutant, indicating either incorrect selection of the conformation of the loop involving residues 140-171, or incorrect substrate positioning. Therefore the interactions between NPP7 and SM are partially defined, and modeled interactions with other substrates have not been supported by experimental results. Further efforts are clearly needed to develop a complete and coherent picture of substrate recognition and discrimination by NPP7.
Lyso-platelet activating factor (lyso PAF), sphingosylphosphorylcholine (SPC), and para-nitrophenyphosphorylcholine (pNPPC, Figure ) are high affinity substrates for NPP7 (Tables , , and , and [10
]). Here we examine the amino acid residues within the NPP7 active site that may be involved in substrate recognition by NPP7. We employed homology modeling to predict protein-substrate interaction points followed by experimental validation of the predictions by mutagenesis and biochemical assays. We used the bacterial Xac
NPP crystal structure [16
] as well as two recently reported NPP2 crystal structures [5
] to generate homology models of NPP7 for this work. Our modeling results reveal a common choline headgroup binding pocket for all substrates. The strong interactions between the cationic choline and the π electrons of the aromatic sidechain of F275 were easily identified by standard docking algorithms. Experimental mutagenesis results are consistent with the predicted choline headgroup positions for all substrates. The hydrophobic tails proved more challenging to place in the complex. Docking algorithms typically cannot exhaustively search the conformations and positions of long and flexible molecules lacking any polar groups that would be involved in directional interactions such as hydrogen bonding. Manual placement was used, and refinements of hydrophobic tail positions by molecular dynamics were required in order to correlate the substrate complex models with the experimental mutagenesis results in the hydrophobic tail region. The molecular dynamics simulations were important to demonstrate the high mobility of the hydrophobic tail positions, as well as to refine the initial complex of sphingomyelin (SM) to explain the different pattern of experimental mutation impacts on activity for this substrate, relative to the other three examined.
Kinetic parameters for hydrolysis of LPC 16:0.
Kinetic parameters for hydrolysis of PAF 16:0.
Kinetic parameters for hydrolysis of SM.
Kinetic parameters for hydrolysis of pNPPC.