title: Role of side-edge site of sphingomyelinase from Bacillus cereus.
abstract: Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan. masa@ph.bunri-u.ac.jp
authors.forename: Masataka
authors.initials: M
authors.lastname: Oda
authors.forename: Masaya
authors.initials: M
authors.lastname: Takahashi
authors.forename: Hideaki
authors.initials: H
authors.lastname: Tsuge
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
authors.forename: Jun
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001409:Bacillus cereus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D010649:Phenylalanine; D001216:Asparagine; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium