E144S active-site mutant of the Bacillus cereus thermolysin-like neutral protease at 2.8 angstrom resolution

The X-ray crystal structure of the Bacillus cereus neutral protease (CNP) active-site mutant E144S, in which the putative general base proposed for the thermolysin-like zinc neutral proteases, Glu144, has been replaced by serine, has been determined to a resolution of 2.8 Angstrom. This represents the first crystal structure of an active-site mutant of a zinc neutral protease. The E144S mutant was crystallized in the hexagonal space group, P6(5)22, with unit-cell dimensions a = b = 76.57, c = 201.91 Angstrom. Although the ligands involved in zinc coordination in the active site are identical to those found in the wildtype protein, the mutation results in a modified environment around the zinc ion; particularly with respect to the water molecules. While the structure of the mutant is similar to that of wild type, its protease activity is reduced to 0.16% that of the wild-type CNP and the protein is virtually resistant to autolysis in the presence of calcium. The lowered protease activity of the mutant is consistent with the role proposed for Glu144 as the general base in the catalysis of thermolysin-like neutral proteases [Matthews (1988). Acc. Chem. Res. 21, 333-340]. We suggest that the residual activity of the E144S mutant arises from a water molecule, which is found within hydrogen-bonding distance of Ser144, acting as a general base in the catalytic function of the mutant.