Glu-53 of Bacillus cereus Sphingomyelinase Acts as an Indispensable Ligand of Mg²⁺ Essential for Catalytic Activity

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Bacillus cereus sphingomyelinase (SMase) is an extracellular hemolysin classified into a group of Mg²⁺-dependent neutral SMases (nSMase). Sequence compartison of bacterial and eukaryotic Mg²⁺-dependent nSMases has shown that several amino acid residues, including Glu-53 of B. cereus SMase, are conserved, suggesting a catalytic mechanism common to these enzymes. Mutational analysis has revealar that hemolytic and SM-hydrolyzing activities are abolished by E53A and E53Q mutations. Only the E53D mutant enzyme partially retains these activities, however, a significant devrease in the apparent k_cat/K_m for SM hydrolysis is observed by this mutation. Mg²⁺activates the wild-type enzyme in a two-step manner, i.e., at least two binding sites for Mg²⁺, high-and low-affinity, are present on the enzyme. The binding affinity of essential Mg²⁺ for the high-affinity site is decreased by the mutation. In addition, the binding affinities of Mn²⁺ and Co²⁺ (substitutes for Mg²⁺) are also decreased. On the contrary, the inhibitory effects of Ca²⁺, Cu²⁺, and Zn²⁺ on SM-hydrolyzing activity are not influenced by the mutation. The results indicate that Glu-53 of B. cereus SMase acts as a ligand for Mg²⁺ and is involved in the high-affinity Mg²⁺-binding site, which is independent of the binding site for inhibitory metals.