Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute^* Faculty of Pharmaceutical Science, Tokushima Bunri University, Japan^** Institute for Health Sciences, Tokushima Bunri University, Japan^*** School of Medicine, Fujita Health University, Japan^****
○Hideo Ago^* Masataka Oda^** Masaya Takahashi^** Hideaki Tsuge^*** Sadayuki Ochi^**** Nobuhiko Katunuma^*** Masashi Miyano^* Jun Sakurai^**

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin (SM) to phosphocholine and ceramide in a divalent metal ion dependent manner. Bc-SMase is a homologue of mammalian neutral SMase (nSMase), and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, aging and apoptosis. Thus Bc-SMase may be a good model for the poorly characterized mammalian nSMase. The metal ion activation of sphingomyelinase activity of Bc-SMase was in the order of Co²⁺ >= Mn²⁺ >= Mg²⁺ >> Ca²⁺ >= Sr²⁺. The first crystal structures of Bc-SMase bound to Co²⁺, Mg²⁺ or Ca²⁺ were determined. The water bridged double divalent metal ions at the center of cleft in both the Co²⁺ and Mg²⁺ bound forms was concluded to be the catalytic architecture require for sphingomyelinase activity. In contrast, the architecture of Ca²⁺ binding at the site showed only one binding site. A further single metal binding site exists at one side edge of the cleft. Based on the highly conserved nature of the residues of the binding sites, the crystal structure of Bc-SMase with bound Mg²⁺ or Co²⁺ may provide a common structural framework applicable to phosphohydrolases belonging to the DNase I like folding superfamily. In addition, the structural features and site directed mutagenesis suggest that the specific b-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound SM substrate.