title: Crystal structure of the human alkaline sphingomyelinase provides insights into substrate recognition.
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.
authors.affiliation: From the Department of Biochemistry and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec H3G 0B1, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Gorelik
authors.affiliation: From the Department of Biochemistry and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec H3G 0B1, Canada.
authors.forename: Fangyu
authors.initials: F
authors.lastname: Liu
authors.affiliation: From the Department of Biochemistry and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec H3G 0B1, Canada.
authors.forename: Katalin
authors.initials: K
authors.lastname: Illes
authors.affiliation: From the Department of Biochemistry and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Quebec H3G 0B1, Canada bhushan.nagar@mcgill.ca.
authors.forename: Bhushan
authors.initials: B
authors.lastname: Nagar
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D002460:Cell Line; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D003902:Detergents / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007313:Insecta; D008823:Micelles; D010767:Phosphorylcholine / Q000737:chemistry; D011485:Protein Binding; D021382:Protein Sorting Signals; D012492:Salts / Q000737:chemistry; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry*; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001647:Bile Acids and Salts; D002412:Cations; D003902:Detergents; D008823:Micelles; D021382:Protein Sorting Signals; D012492:Salts; D010767:Phosphorylcholine; D014443:Tyrosine; C508908:ENPP7 protein, human; D013108:Sphingomyelin Phosphodiesterase
keywords: ENPP7; NPP7; alk-SMase; bile acid; choline; micelle; sphingolipid; sphingomyelinase