Family S45


Summary Holotypes Alignment Tree Genomes Structure Literature

Summary for family S45

Family type peptidaseS45.001 - penicillin G acylase precursor (Escherichia coli), MEROPS Accession MER0003306 (peptidase unit: 290-846)
Content of familyPeptidase family S45 contains the self-cleaving precursor proteins of N-terminal nucleophile acylases.
History Identifier created: MEROPS 5.00 (20 April 2000)
Penicillin G acylase precursor (S45.001) and cephalosporin acylase precursor (S45.002, more precisely termed glutaryl-7-aminocephalosporanic acid acylase) are self-cleaving proteins that spontaneously form the heterodimeric subunits of the mature acylases. They are not known to hydrolyse peptide bonds in other substrates, although they may have the capacity to do so (see below).
Catalytic typeSerine
Active site residuesS290 
Active siteThe primary catalytic residues in both penicillin G acylase precursor and cephalosporin acylase precursor are serine residues (see the Alignment) in which the hydroxyl is the nucleophile in catalysis, and its amino group serves as a general base (Duggleby et al., 1995; Kim et al., 2003). It has been reported that His and Glu residues in the beta chain of the Pseudomonas cephalosporin acylase contribute to catalysis (Kim et al., 2003; Mao et al., 2004), but whether there is a catalytic triad that is in any way analogous to those in families S1 and S8, for example, has yet to be established. In penicillin acylase, there is evidence for an additional cleavage mediated by a threonine residue a few residues from the catalytic serine (Hewitt et al., 2000). The Ser of the active site of the peptidase becomes the key residue of the active site of the mature acylase, also.
Activities and specificitiesFor both penicillin G acylase precursor and cephalosporin acylase precursor the maturation reaction involves two proteolytic cleavages. First, the bond on the amino side of the catalytic serine is cleaved in an intramolecular reaction, and then there is an intermolecular cleavage of a bond further toward the N-terminus. This second cleavage liberates a 'linker' or 'spacer' peptide. The remaining N-terminal part of the chain then forms the alpha subunit and the C-terminal part the beta subunit that interact non-covalently to form the alpha-beta dimer of the catalytically active acylase (which is commonly a dimer of these dimers). It has been shown that the maturation requires the loss of the linker peptide, which contains 54 residues in penicillin G acylase precursor but only 9 residues in cephalosporin acylase precursor (Hewitt et al., 2000; Kim et al., 2003). There is no report that the mature acylases retain peptidase activity, but if the substrate specificity of the second, intermolecular cleavage were not strict, there would appear to be the possibility of action on some other protein or peptide.
InhibitorsIn penicillin acylase precursor, the newly formed N-terminal Ser was reactive with PMSF, which inactivated the acylase (Duggleby et al., 1995); it was not shown in this study whether the second peptidase reaction was affected by PMSF. Neither the cephalosporin acylase precursor nor its mature enzyme was found to be affected by DFP, PMSF or APMSF (Park, 2004). The carbamylation of the alpha-amino group of the catalytic Ser in cephalosporin acylase precursor with cyanate prevented the second maturation cleavage and the formation of active acylase (Lee et al., 2000).
Molecular structureStructures are available for both penicillin G acylase precursor (Hewitt et al., 2000) and cephalosporin acylase precursor (Kim et al., 2003). The C-terminal parts of the molecules adopt folds typical for clan PB. The space around the N-terminal, serine nucleophile, which becomes the active site cleft in the mature enzymes, is filled by the linker peptide in the precursors. The mature cephalosprorin acylase is an (alpha, beta)2) heterotetramer formed by interaction of a pair of the autolytically-generated heterodimers.
Basis of clan assignmentProtein fold of the peptidase unit for members of this family resembles that of archaean proteasome subunit B, the type example of clan PB.
Distribution of family Bacteria details  
Archaea details  
Protozoa details  
Fungi -  
Plants details  
Animals details  
Viruses -  
Biological functionsBoth penicillin G acylase precursor and cephalosporin acylase precursor are periplasmic proteins, synthesised with signal peptides that are removed before the maturation cleavages described above. One could speculate that the mature enzymes confer on their host organisms resistance to antibiotics in their natural environments.
Pharmaceutical and biotech relevanceBoth of the enzymes in family S45 are of great importance in the commercial production of antibiotics. They are used to remove the naturally-occurring side chain (e.g. glutarate) from the antibiotic precursors following synthesis in microbial culture to allow the chemical addition of one of a variety of artificial side chains that lead to the many variants of the penicillin and cephalosproin antibiotics (Barends et al., 2004). The enzymes are the subjects of mutagenic studies to improve their effectiveness in the commercial processes.
Statistics for family S45Sequences:2584
Identifiers with PDB entries:5
Downloadable files Sequence library (FastA format)
Sequence alignment (FastA format)
Phylogenetic tree (Newick format)
Peptidases and Homologues MEROPS ID Structure
penicillin G acylase precursorS45.001Yes
cephalosporin acylase precursorS45.002Yes
cephalosporin C acylase precursorS45.003Yes
acyl-homoserine lactone acylaseS45.004-
aculeacin A acylaseS45.005Yes
family S45 non-peptidase homologuesnon-peptidase homologue-
family S45 unassigned peptidasesunassignedYes