(a) The Ca2+-binding site Ca3 in the N-terminal domain of the P. polymyxa neutral protease Gentlyase. Water molecules coordinating the Ca2+ ion (yellow) are shown as red spheres. (b) The Ca3 site in the prototypic neutral protease thermolysin from B. thermoproteolyticus (PDB entry 3fvp
). (c, d) Superimposition of the neutral protease Gentlyase (yellow) with thermolysin (green) shows that the deletion in Gentlyase does not affect the Ca3 site. However, the backbone conformation and side-chain orientation of Asn59 adjacent to the deletion is affected. The corresponding Phe63 is reported to confer thermostability on thermolysin or B. stearothermophilus neutral protease by stabilizing the N-terminal β-sheet through hydrophobic interactions with the side chain (Van den Burg et al., 1994). Owing to the three-residue deletion after Asn59 these stabilizing hydrophobic contacts cannot be formed in Gentlyase, even if the thermolysin Phe63 were not changed to Asn59 in Gentlyase.