Glu-53 of Bacillus cereus sphingomyelinase acts as an indispensable ligand of Mg2+ essential for catalytic activity

Bacillus cereus sphingomyelinase (SMase) is an extracellular hemolysin classified into a group of Mg2+-dependent neutral SMases (nSMase). Sequence comparison 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 revealed 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 decrease 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.