Understanding the Molecular Activity of Alkaline Sphingomyelinase (NPP7) by Computer Modeling

Abstract

The enzymes in the nucleotide pyrophosphatase/phosphodiesterase (NPP) family have various substrates such as nucleotides, phospholipids, and sphingolipids. The substrate specificity in relation to their structures is largely unknown because no mammalian NPP complex has been crystallized. NPP7, also called alkaline sphingomyelinase (alk-SMase), is a NPP family member that may have important implications in carcinogenesis and cholesterol absorption. The sequence of NPP7 is 36% similar to that of the closest NPP member, but NPP7 has no activity against nucleotides. In this work, we predict the three-dimensional structure of NPP7 by homology modeling using a recently crystallized NPP from bacteria. Using the model, we studied the substrate specificity of the enzyme by docking. The model generated explains the functional changes in previous mutagenesis studies and rationalizes the structural basis for the lack of activity toward nucleotides. An effort to shift the substrate specificity from sphingomyelin (SM) to nucleotide was not successful but revealed a site-directed mutation that increased activity toward SM. In conclusion, this is the first study to predict the structure of a mammalian NPP and its substrate specificity by molecular modeling. The information may be helpful in understanding the functional differences of NPP members.

Computational identification and experimental characterization of substrate binding determinants of nucleotide pyrophosphatase/phosphodiesterase 7

“…This interaction site was previously described by Duan, et al , in their modeling study on NPP7. [15] They found that an F275G mutant showed almost no sphingomyelinase activity, consistent with our reduced activity of the F275A mutant. Perhaps surprisingly, an alignment of the mammalian NPP enzymes shows remarkable variability in the residue types that align with F275.…”

“…Due to the much smaller gap-aligned segments in the 2GSO-based model of hNPP7, this model was used in all docking studies with substrates. Two prior modeling studies of hNPP7 also used a crystallographic structure of the bacterial NPP as the modeling template [15,24], although one of the studies was published prior to the availability of the NPP2 crystal structures [15]. The insertion of residues in the NPP7 sequence within the segment interacting with LPA in NPP2 limits the utility of the NPP2 crystal structures as templates even though they share a common substrate with NPP7.…”

“…Mutation of F141 (yellow) to serine was reported by Duan, et al . to substantially reduce sphingomyelinase activity of NPP7 [15]. Distance between F141 and the palmitoyl tail is 2.8 Å.…”

“…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.…”

Site-Directed Mutagenesis Maps Interactions That Enhance Cognate and Limit Promiscuous Catalysis by an Alkaline Phosphatase Superfamily Phosphodiesterase

“…However, whereas other members of the AP superfamily transfer an unsubstituted phosphoryl group (−PO 3 2− ) and donate hydrogen bonds to both of the nonbridging phosphoryl oxygen atoms of monoester substrates, NPP transfers a substituted phosphoryl group (−PO 2 OR′ − ) and has a substituent binding pocket that interacts with the R′ substituent attached to one of the phosphoryl oxygen atoms of its diester substrates (Figure 1). 17,22–24 This pocket functionally replaces one set of hydrogen bonds that provide favorable interactions with monoester substrates in the monoesterase members of the superfamily. Depending on the architecture of the pocket, substrates for enzymes in the NPP family can be nucleotides or lipids, such as choline phosphoesters, and sphingomyelin.…”