| Name | Peptidase family M4 (thermolysin family) |
|---|
| Family type peptidase | M04.001 - thermolysin (Bacillus thermoproteolyticus), MEROPS Accession MER0001026 (peptidase unit: 233-548) |
| Content of family | Peptidase family M4 contains mainly metalloendopeptidases. |
| History |
Identifier created: Biochem.J. 290:205-218 (1993)
Thermolysin (M04.001) was one of the first metallopeptidases to be sequenced (Titani et al., 1972) and one of the first to have its tertiary structure determined (Matthews et al., 1972). |
| Catalytic type | Metallo |
| Active site residues | H374 E375 H378 E398 H463 |
| Active site | All peptidases in the family bind a single, catalytic zinc ion. As in many other families of metallopeptidases, there is an HEXXH motif, in which the histidines are zinc ligands and the glutamate (Glu375) is an active site residue. This common motif was refined by Jongeneel et al., 1989. The Jongeneel consensus identifies most mono-catalytic zinc metallopeptidases from a number of families. The zinc is bound by a glutamate (Glu398), 20-33 residues C-terminal to the HEXXH motif. Metallopeptidases in which the zinc is bound by HEXXH plus Glu are known as 'Glu-zincins'. A zinc ion is tetrahedrally co-ordinated, and the fourth ligand is activated water which forms the nucleophile in catalysis. The mechanism of catalysis by thermolysin and its homologues is controversial, especially as regards which amino acids take part in catalysis. For example, Glu375 was originally considered a proton donor, but Mock & Stanford, 1996 consider it an electrophile. Other residues found to be essential for catalysis are Tyr389, Asp402, Asp458 and His463. It is His463 that Mock & Stanford propose to be the proton donor and general base, and Asp458 is thought to orientate the imidazolium ring of His463 in much the same way as an aspartate does in the catalytic triad of serine peptidases. Asp458 is not strictly conserved in the family, however. Tyr389 may act as a general acid and help to position the nucleophilic water molecule. Asp402 is completely conserved amongst all active members of the family and forms a hydrogen bond with His463 (Argos et al., 1978); the Glu-Xaa-Xaa-Xaa-Asp is a second useful motif for detecting members of the family. |
| Activities and specificities | Most members of the family are endopeptidases active at neutral pH. Proteins and peptides are degraded with a preference for cleavage of Xaa+Yaa, in which Xaa is a hydrophobic residue and Yaa is Leu, Phe, Ile, or Val. Furanacryoyl-Gly-Leu-amide is a typical synthetic substrate. |
| Inhibitors | Reversible inhibitors include 1,10-phenanthroline and EDTA. The naturally occurring phosphoramidate phosphoramidon (from Streptomyces tanashiensis) inhibits with a Ki of 30nM (Suda et al., 1973). Protein inhibitors include Streptomyces metalloproteinase inhibitor (I36.001) and inducible metalloproteinase inhibitor from the moth Galleria mellonella caterpillar (I08.006). |
| Molecular structure | Thermolysin has a two-domain structure with the active site between the domains. The N-terminal domain includes a distinctive six-strand beta sheet with two helices, one of which carries the HEXXH zinc-binding motif. The C-terminal domain, which is unique for the family, is predominantly helical and carries the third zinc ligand. Thermolysin is the type-example of clan MA. Other families in clan MA, such as M10 (M10) and M12 (M12), share a similar core structure to the thermolysin N-terminal domain, but the C-terminal domains are unrelated. |
| Clan | MA |
| Subclan | MA(E) |
| Basis of clan assignment | Type family of clan MA. |
| Biological functions | Most members of the family are secreted enzymes that degrade extracellular proteins and peptides for bacterial nutrition, especially prior to sporulation . They have N-terminal propeptides that are autocatalytically removed. It has been shown that the propeptide assists folding of the protein (O"Donohue & Beaumont, 1996). In pseudolysin (M04.005) the propeptide is cleaved in the periplasm but remains associated with the mature peptide and this noncovalent complex is inactive. Translocation to the medium requires a complex protein export apparatus known as Xcp. It has recently been suggested that the propeptide contains a peptidase inhibitor domain known as 'pepsy' (Yeats et al., 2004). Pseudolysin is the major virulence factor of Pseudomonas aeruginosa septicaemia, causing tissue damage (especially in the lung where elastin is degraded) and may compromise the immune system by degradation of immunoglobulins, complement components and serpins. Msp protease (M04.005) from Legionella may have a role in the virulence of Legionaire"s disease (Sahney et al., 2001). Vibriolysin (M04.004) from Vibrio cholerae is a haemagglutinin that has been shown to affect intracellular tight junctions by degrading occludin (Wu et al., 2000). |
| Pharmaceutical and biotech relevance | Thermolysin is widely used as a nonspecific proteinase to obtain fragments for peptide sequencing. It has also been used as a peptide synthetase and in production of the artificial sweetener aspartame (Ooshima et al., 1985). |
| Statistics for family M4 | Sequences: | 6399 |
| Identifiers: | 23 |
| Identifiers with PDB entries: | 6 |
| Downloadable files |
Sequence library (FastA format) |
| Sequence alignment (FastA format) |
| Phylogenetic tree (Newick format) |
