Abstract
Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase (alk-SMase, NPP7, ENPP7). alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules. NPP7 is the only paralog that can cleave SM, and its activity requires the presence of bile salts, a class of physiological anionic detergents. To elucidate the mechanism of substrate recognition, we determined the crystal structure of human alk-SMase in complex with phosphocholine, a reaction product. Although the overall fold and catalytic center are conserved relative to other NPPs, alk-SMase recognizes the choline moiety of its substrates via an NPP7-specific aromatic box composed of tyrosine residues. Mutational analysis and enzymatic activity assays identified features on the surface of the protein-a cationic patch and a unique hydrophobic loop-that are essential for accessing SM in bile salt micelles. These results shed new light on substrate specificity determinants within the NPP enzyme family.
Highlights
Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase. alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules
The overall fold and catalytic center are conserved relative to other NPPs, alk-SMase recognizes the choline moiety of its substrates via an NPP7-specific aromatic box composed of tyrosine residues
Despite the rather low sequence identity of ϳ30% between alk-SMase and the other NPPs, secondary structure elements are overall conserved (Fig. 1B). alk-SMase contains no disulfide bonds and possesses five N-linked glycosylation sites, all required for full activity [25] and visible in the structure (Fig. 1A), three of which are located on the face of the enzyme harboring the active site
Summary
Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase (alk-SMase, NPP7, ENPP7). alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules. Mutational analysis and enzymatic activity assays identified features on the surface of the protein—a cationic patch and a unique hydrophobic loop—that are essential for accessing SM in bile salt micelles. These results shed new light on substrate specificity determinants within the NPP enzyme family. Alk-SMase is a member of the (ecto)nucleotide pyrophosphatase/phosphodiesterase protein family (NPP), a group of seven extracellular enzymes that act on diverse substrates to carry out various functions [11]. NPP4 participates in the activation of platelet aggregation via cleavage of diadenosine triphosphate [13] In contrast to these enzymes acting on extracellular nucleotides involved in purinergic signaling, other NPP members degrade phospholipid substrates. Structural and functional analysis identified features on the surface of the protein essential for accessing SM in bile salt micelles
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