Structure Of Thermolysin Research Articles

Structure of gentlyase, the neutral metalloprotease of Paenibacillus polymyxa.

Gentlyase is a bacterial extracellular metalloprotease that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases. The structure of thermolysin has been known since 1972 and that of Bacillus cereus neutral protease since 1992. However, the structure determination of other Bacillus neutral proteases has been hindered by their tendency to cannibalistic autolysis. High calcium conditions that allow the concentration and crystallization of the active Gentlyase metalloprotease without autoproteolysis were identified using thermal fluorescent shift assays. X-ray structures of the protease were solved in the absence and in the presence of the inhibitor phosphoramidon at 1.59 and 1.76 Å resolution, respectively. No domain movement was observed upon inhibitor binding, although such movement is thought to be a general feature of the thermolysin-like protease family. Further analysis of the structure shows that the observed calcium dependency of Gentlyase stability may arise from a partly degenerated calcium site Ca1-2 and a deletion near site Ca3.

Thermolysin in the absence of substrate has an open conformation.

The bacterial neutral proteases have been proposed to undergo hinge-bending during their catalytic cycle. However, in thermolysin, the prototypical member of the family, no significant conformational change has been observed. The structure of thermolysin has now been determined in a new crystal form that for the first time shows the enzyme in the absence of a ligand bound in the active site. This is shown to be an 'open' form of the enzyme. The relative orientation of the two domains that define the active-site cleft differ by a 5 degrees rotation relative to their positions in the previously studied ligand-bound 'closed' form. Based on structural comparisons, kinetic studies on mutants and molecular-dynamics simulations, Gly78 and Gly135-Gly136 have previously been suggested as two possible hinge regions. Comparison of the 'open' and 'closed' structures suggests that neither of the proposed hinge regions completely accounts for the observed displacement. The concerted movement of a group of side chains suggested to be associated with the hinge-bending motion is, however, confirmed.

Molecular Modeling of the Active Site of Endothelin-Converting Enzyme

Endothelin-converting enzyme (ECE) is a member of the zinc metalloproteinase family. It is much more specific in its protease activity than the bacterial metalloprotease thermolysin; we aim to construct a model of its active site to help to explain these differences. We aligned the sequence of human ECE with those of human neprilysin (which is 39% identical to ECE) and thermolysin. Residues believed to be important for inhibitor binding were assigned from the alignment and by analogy with structural and functional studies of these enzymes. These included a conserved IGG motif N-terminal to the zinc-binding HExxH motif, and a tyrosine residue that may be analogous to Y157 of thermolysin. We have used the program O to build a model of the active site of ECE based on the crystal structure of thermolysin.

Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated.

A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.

Thermal stability of homologous neutral metalloendopeptidases in thermophilic and mesophilic bacteria: structural considerations.

Thermolysin and neutral protease A are neutral metalloendopeptidases having similar specificity, molecular weight, metal content, and amino acid composition. Thermolysin, derived from the thermophilic organism Bacillus thermoproteolyticus, is heat inactivated at about 84 degrees whereas neutral protease A, derived from the mesophilic organism Bacillus subtilis, is inactivated at about 59 degrees. Structural analyses reveal that the two enzymes are homologous. Of the 326 residues of neutral protease A, 171 have been placed in sequence and 49% of these have been found in identical loci in thermolysin. These include many of the residues corresponding to the active site of thermolysin. The sensitivity of both enzymes to thermal inactivation is dependent upon the presence of calcium and neutral protease appears to bind less calcium than thermolysin. Structural data indicate that many of the ligands associated with calcium sites 1 and 2 (double site of thermolysin) are present in neutral protease and that calcium site 4 cannot exist in neutral protease. The structural homology and functional analogy of these two proteins support the concept that they have similar conformations. The known structure of thermolysin is used as a model to discuss structural differences which might be related to thermal stability.

Structure of Thermolysin

Results of the chemical and X-ray analyses are combined to locate the active site and the calcium binding sites.