InterPro: IPR013856 Peptidase M4, thermolysin

In the MEROPS database peptidases and peptidase homologues are grouped into clans and families. Clans are groups of families for which there is evidence of common ancestry based on a common structural fold:

• Each clan is identified with two letters, the first representing the catalytic type of the families included in the clan (with the letter 'P' being used for a clan containing families of more than one of the catalytic types serine, threonine and cysteine). Some families cannot yet be assigned to clans, and when a formal assignment is required, such a family is described as belonging to clan A-, C-, M-, S-, T- or U-, according to the catalytic type. Some clans are divided into subclans because there is evidence of a very ancient divergence within the clan, for example MA(E), the gluzincins, and MA(M), the metzincins.
• Peptidase families are grouped by their catalytic type, the first character representing the catalytic type: A, aspartic; C, cysteine; G, glutamic acid; M, metallo; S, serine; T, threonine; and U, unknown. The serine, threonine and cysteine peptidases utilise the amino acid as a nucleophile and form an acyl intermediate - these peptidases can also readily act as transferases. In the case of aspartic, glutamic and metallopeptidases, the nucleophile is an activated water molecule.

In many instances the structural protein fold that characterises the clan or family may have lost its catalytic activity, yet retain its function in protein recognition and binding.

Metalloproteases are the most diverse of the four main types of protease, with more than 50 families identified to date. In these enzymes, a divalent cation, usually zinc, activates the water molecule. The metal ion is held in place by amino acid ligands, usually three in number. The known metal ligands are His, Glu, Asp or Lys and at least one other residue is required for catalysis, which may play an electrophillic role. Of the known metalloproteases, around half contain an HEXXH motif, which has been shown in crystallographic studies to form part of the metal-binding site [1]. The HEXXH motif is relatively common, but can be more stringently defined for metalloproteases as 'abXHEbbHbc', where 'a' is most often valine or threonine and forms part of the S1' subsite in thermolysin and neprilysin, 'b' is an uncharged residue, and 'c' a hydrophobic residue. Proline is never found in this site, possibly because it would break the helical structure adopted by this motif in metalloproteases [1].

This group of metallopeptidases constitutes the MEROPS peptidase family M4 (thermolysin family, clan MA(E)). The protein fold of the peptidase domain of thermolysin, is the type example for members of the clan MA. The thermolysin family is composed only of secreted eubacterial endopeptidases. The zinc-binding residues are H-142, H-146 and E-166, with E-143 acting as the catalytic residue. Thermolysin also contains 4 calcium-binding sites, which contribute to its unusual thermostability. The family also includes enzymes from a number of pathogens, including Legionella and Listeria, and the protein pseudolysin, all with a substrate specificity for an aromatic residue in the P1' position. Three-dimensional structure analysis has shown that the enzymes undergo a hinge-bend motion during catalysis. Pseudolysin has a broader specificity, acting on large molecules such as elastin and collagen, possibly due to its wider active site cleft [1].