(a) Sequence alignment between the neutral proteases from Paenibacillus polymyxa (Gentlyase), Bacillus thermoproteolyticus (thermolysin), B. cereus (UniProt ID Q63G45_BACCZ) and B. stearothermophilus (NPRT_GEOSE). The numbering shown corresponds to the mature proteases. Residues liganding Ca2+ in Ca1–2, Ca3 and Ca4 are boxed in red, blue and black, respectively. Acidic, basic, polar and nonpolar residues are coloured red, blue, magenta and black, respectively. (b) Superposition of the overall structure of P. polymyxa neutral protease (N-terminal domain in cyan and C-terminal domain in blue) with that of thermolysin from B. thermoproteolyticus (green; PDB entry 3fvp
). Calcium-binding sites Ca1–2, Ca3 and Ca4 are discernible by the yellow Ca2+ ions and are labelled 1, 3 and 4, respectively. The active site is marked by the grey Zn2+ ion. There is overall high structural similarity, except for the two helices close to the calcium-binding sites Ca1–2 and Ca3, which are shortened by one turn in P. polymyxa neutral protease (black arrows) owing to the two sequence deletions.