Amidohydrolases include glutamine PRPP amidotransferases (GATs), penicillin acylases/amidases (such as penicillin G acylases or PGAs and penicillin V acylases or PVAs), the 20S proteasome beta subunit (PRO), class II glutamine amidotransferases, glycosyl asparaginases (GAs), and also cephalosporin acylases, such as CAD, a class I cephalosporin acylase. The importance of Ntn hydrolases resides in the biosynthesis of beta-lactam antibiotics such as cephalosporin and penicillin. All enzymes in this superfamily share an unusual fold in which the nucleophile and other catalytic groups occupy equivalent sites. This fold provides both the capacity for nucleophilic attack and the possibility of autocatalytic processing. Posttranslational autoproteolysis is a mechanism used to activate many proteins via self-catalysed peptide bond rearrangements, which play an essential role in a wide variety of biological processes. They include activation cascades such as blood coagulation and fibrinolysis, cell death, embryonic development, protein targeting and degradation, viral protein processing, and zymogen activation.

The N-terminal nucleophile aminohydrolase (Ntn) domain consists of two catalytic groups - a proton donor and a nucleophile. This superfamily consists of the nucleophile catalytic domain or Ntn hydrolase domain. The amino acid residue (cysteine in GAT, serine in penicillin acylase, and threonine in proteasome) that initiates the nucleophilic attack at the carbonyl group is located at the N-terminus of this domain. The Ntn fold domain comprises four layers of alpha-helices and beta-sheets in an alpha-beta-beta-alpha sandwich structural motif. Ntn hydrolases have divergent sequences, but share a common enzyme mechanism.

A well-characterised enzyme containing this domain is CAD, a cephalosporin acylase from Pseudomonas diminuta. It consists of an alpha-subunit with 169 residues and a beta-subunit with 520 residues. The enzyme is described as a bowl-shaped molecule with two knobs attached to the bowl, facing each other. The alpha-beta-beta-alpha motif is the central part of the molecule. Ser-1, the N-terminal residue of the beta subunit is invariant and known to play a critical role in both autoproteolytic activation and enzyme catalysis.

Pfam clan [PfamClan:CL0052], Pfam family PFAM:PF01804, INTERPRO:IPR029055,PMID:7477383,PMID:11601852,PMID:11080627,PMID:16446446,PMID:23373797