Abstract
Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin 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 Co2+ > or = Mn2+ > or = Mg2+ >> Ca2+ > or = 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 the cleft in both the Co2+- and Mg2+-bound forms were concluded to be the catalytic architecture required 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 beta-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound sphingomyelin substrate.
Highlights
The catalytic mechanism of the sphingomyelin hydrolytic activity remains to be elucidated in atomic detail, as there are no crystal structures of SMase in complex with the essential divalent metal ions
The sphingomyelin hydrolytic activity of Bc-SMase is believed to proceed in the manner of acid base catalysis, in which His-296 is proposed to generate an activated water and the essential Mg2ϩ ion at Glu-53 is suggested to stabilize a negatively charged transition state
In this study we propose that the water-bridged double divalent metal ions are essential in the hydrolytic activity of Bc-SMase based on the JUNE 9, 2006
Summary
Expression and Purification—Bc-SMase was overexpressed in Bacillus subtilis ISW1214 transformed with the plasmid vector, pHY300PLK, carrying cDNA of Bc-SMase cloned from B. cereus IAM 1029. The reaction mixture containing the enzyme at various concentrations, 3% (w/v) sheep erythrocytes, 20 mM Tris-HCl buffer (pH 7.5), 3 mM MgCl2, and 0.9% (w/v) NaCl, was incubated for 30 min at 37 °C and centrifuged at 500 ϫ g for 3 min in order to prepare the test aliquot. The SM liposome solution containing 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, and 0.9% (w/v) NaCl was incubated with Bc-SMase for 30 min at 37 °C. The crystallization drop for the calcium acetate-bound form was prepared by mixing equal volumes of protein solution (10 mg/ml Bc-SMase in 20 mM Tris-HCl (pH 7.0)) and reservoir solution (18% (w/v) polyethylene glycol 8000, 0.2 M Ca(OCOCH3), and 0.1 M sodium cacodylate (pH 6.5)). The diffraction images of the crystal of the magnesium-bound form to 1.80 Å resolution were col-
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