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Asparagine peptide lyases are a catalytic type of proteolytic enzymes, not peptidases or hydrolases, that self-cleave peptide bonds in proteins in which asparagine serves as the nucleophilic amino acid at the active site. The discovery of “asparagine peptide lyases” indicates that not all peptide bond cleavage occurs by hydrolysis.

The asparagine can be induced to attack its own carbonyl bond leading to the formation of a succinimide ring and peptide bond cleavage. Usually, a second acidic residue is required. The term “asparagine peptide lyase”, describes this catalytic activity. All asparagine peptide lyases are self-cleaving proteins, with cleavage occurring in cis.

There are six families of viral coat proteins (including families N1 and N2, previously considered aspartic peptidases), three families of intein-containing proteins and two families of bacterial autotransporters.

Classification
The MEROPS protease classification system counts 6 clans that includes 10 families,  based on sequence similarity in which cleavage is thought to be catalyzed by an asparagine. Each family includes proteolytic enzymes with homologous sequences and the same catalytic type.

Clans are groups of proteolytic enzymes families with related structures, where catalytic type is not conserved.

Asparagine peptide lyases have been classified in ten families. There are five families of viral coat proteins (N1, N2, N8, N7 and N5), two families of autotransporter proteins (N4 and N6) and three families of intein-containing proteins (N9, N10 and N11).

Autotransporter proteins
Autotransporters are virulence factors secreted by pathogenic, Gram-negative bacteria, an example being Tsh (S06.003) which is a peptidase secreted by Escherichia coli. It is synthesized as a precursor with a signal peptide and a large C-terminal propeptide (Stathopoulos et al., 1999). The C-terminal propeptide forms a pore in the outer membrane through which the peptidase domain can pass. Autolytic cleavage then occurs at Asn1100, releasing the peptidase.

The secretion of a protein in a Gram-negative bacterium involves passage through the periplasm and cell membranes. Presence of a signal peptide ensures the protein reaches the periplasm, but to allow it pass through the outer cell membrane a different mechanism is needed. The tsh peptidase from E. coli is synthesized as a precursor with a large C-terminal propeptide that acts as an autotransporter. The structure shows that this domain forms a pore in the outer membrane through which the N-terminus its released to the exterior by autolytic cleavage at Asn1100.

Family N4: secreted proteins from enterobacteria where domains homologous of the C-terminal autotransporter domain have been found.

Family N6: proteins involved in type III protein secretion system. This system secretes proteins directly into host cells by an injectisome, a hollow tube that penetrates into the host cell down which unfolded proteins can pass. The FlhB protein from E. coli is part of the basal body which sits in the bacterial cell membrane opening to the cytoplasm and its self-cleaving at an Asn + Pro-Thr-His motif controls the change from one protein to another.

Viral coat proteins
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Family N1 nodavirus peptide lyase (flock house virus)

Family N2 tetravirus coat protein (Nudaurelia capensis omega virus)

Family N8 poliovirus capsid CP0-type self-cleaving protein (human poliovirus 1)

Family N5 picobirnavirus self-cleaving protein (Human picobirnavirus)

Family N7 reovirus type 1 coat protein (Mammalian orthoreovirus 1)

Clan PD (N)
Family N9 intein-containing V-type proton ATPase catalytic subunit A (Saccharomyces cerevisiae)

Family N10 intein-containing replivative DNA helicase precursor (Synechocystis sp. PCC 6803)

Family N11 intein-containing chloroplast ATP-dependent peptide lyase (Chlamydomonas eugametos)

Mechanism
All of the asparagine peptide lyases perform only self-cleavages, with cleavage occurring immediately C-terminal to the active site asparagine. Cleavage occurs when this asparagine forms a succimide ring, which happens when an second active site residue (an aspartate or a glutamate) is brought into close proximity. Cleavage is intra-molecular in cis.

Inhibition
No inhibitors are known.