Zinc protease of Bacillus subtilis var. amylosacchariticus: construction of a three-dimensional model and comparison with thermolysin.

The active site structure of the Zn-containing neutral protease from Bacillus subtilis var. amylosacchariticus (BANP) was predicted by computer-aided modeling on the basis of the three-dimensional structure of thermolysin (TLN). As expected from the high homology in amino acid sequence of the two enzymes, the overall folding of BANP was very similar to that of TLN. Glu144, Tyr158, and His228 of BANP were located near the active site Zn ion, to which three amino acid residues, His143, His147, and Glu167, were coordinated. This model is supported by the previous results that chemical modifications of Tyr158 and photooxidation of His228 of BANP markedly affect the proteolytic activity of the enzyme. Interestingly, BANP was found to be significantly less sensitive to metalloprotease inhibitors such as phosphoramidon and talopeptin. From a comparison of the enzyme-inhibitor complex models between BANP and thermolysin, it is suggested that replacement of Thr129 in TLN by Phe130 in BANP is related to difference in inhibitor sensitivity between BANP and TLN.

The structure of neutral protease from Bacillus cereus at 0.2-nm resolution.

The crystal structure of the neutral protease from Bacillus cereus has been refined to an R factor of 17.5% at 0.2-nm resolution. The enzyme, an extracellular metalloendopeptidase, consists of two domains and binds one zinc and four calcium ions. The structure is very similar to that of thermolysin, with which the enzyme shares 73% amino-acid sequence identity. The active-site cleft between the two domains is wider in neutral protease than in thermolysin. This suggests the presence of a flexible hinge region between the two domains, which may assist enzyme action. The high-resolution analysis allows detailed examination of possible causes for the difference in thermostability between neutral protease and thermolysin.