BACILLUS CEREUS NEUTRAL PROTEASE G197D AND E144S MUTANT STRUCTURES.
S.A. Litster and P.W. Codding, CNP) were developed and the structures determined. Here we present the crystal structures of the G197D and E144S mutants of CNP, at 3.0Å and 2.8Å resolution, respectively. A comparison of the structures and how they relate to the thermal stability and hydrolyase activity of the enzyme will be made. The G197D structure is novel in that it contains only three calcium ions, with the missing calcium ion being Ca(II); the ion thought to bind cooperatively along with Ca(I) to form the double calcium binding site in native CNP and thermolysin. The second structure, that of the inactive E144S mutant, the Glu to Ser mutation reduces the protease activity of the enzyme to 0.16% that of wild type and represents the first crystal structure of an active site mutant of a neutral protease. The mutant structure reveals a modified environment around the catalytic zinc ion and suggests a major role for bound water molecules. The mutants crystallize in the hexagonal space group P6(sub5)22 which is isomorphous with wild type crystals.