|Family type peptidase||N09.001 - intein-containing V-type proton ATPase catalytic subunit A (Saccharomyces cerevisiae), MEROPS Accession MER0180658 (peptidase unit: 284-493, 643-738)|
|Content of family||Family N9 includes self-cleaving proteins|
Identifier created: MEROPS 9.4 (31 January 2011)|
An intein is a polypeptide insert into another protein (the extein) which is able to release itself from the host protein and splice the two portions of the extein together. Thus two proteins are generated from one: the extein and the intein. The intein is a multidomain protein, usually containing the domain responsible for cleavage and splicing divided into two portions, one at the N-terminus and one at the C-terminus, with an endonuclease domain known as a 'homing endonuclease' in the middle. The homing endonuclease recognizes a rare nucleotide sequence which it cleaves to leave a staggered break, usually of four bases. The staggered break allows the cleaved DNA to pair with the intein gene so that when the cell"s repair mechanism mend the break, the intein gene is used as a template with the effect that the intein gene is copied. The intein gene has replicated itself in a non-Mendelian manner, akin to that of some introns and transposons, and an intein gene can be considered an example of parasitic DNA. The burden on the host is light because the homing endonuclease recognition sequence is rare, and it is unusual for a genome to contain more than one intein, and because cleavage occurs within the recognition sequence, and once a gene has acquired an intein gene it cannot acquire another. Replication of the intein gene can occur during meiosis in eukaryote cells, and in prokaryote cells can occur by horizontal gene transfer (Raghavan & Minnick, 2009). The nature of the extein is irrelevent except that its gene must include the endonuclease recognition sequence. In the case of the VMA intein (N09.001) from Saccharomyces cerevisiae, the intein is an endonuclease (endonuclease PI-SceI), and the exteins, once spliced, become catalytic subunit A of the V-type proton ATPase.
|Active site residues||C,S,T284 N737 T,C,S738 |
|Active site||Three residues are involved in activity, the first (Cys) and last (Asn) residues of the intein, and the first residue of the second portion of the extein (Cys, Ser or Thr). Each of these acts as a nucleophile in each of the three reactions that occur to excise the intein and splice the remaining portions of the extein. The penultimate residue of the intein is often His, and this may be important for the cyclization of the Asn and thus cleavage of the bond between the Asn and the second portion of the extein.|
|Activities and specificities||The release of the intein from the extein is a self-cleaving event. Two peptide bond cleavages occur, at the residue immediately preceding the first residue of the intein and following the last residue. The first residue of the intein is Cys and the last residue of the intein is Asn. The first residue of the second portion of the extein must be Cys, Ser or Thr. The thiol on the side chain of the first residue of the intein attacks the carbonyl carbon of the preceding amino acid to generate a thiolester intermediate. Then transesterification occurs where the first residue of the second portion of the extein attacks the thioester. This results in a branched intermediate where the first portion of the extein is transfered to the side chain of the first residue of the second portion and the peptide bond between the first portion of the extein and the first residue of the intein is broken. The branched intermediate thus has two N-termini. Then the last residue of the intein, which is Asn, cyclizes to form a succinimide ring, which results in the breakage of the peptide bond between the Asn and the first residue of the second portion of the extein. The intein is thus released. The thioester bond in the extein then rapidly undergoes an acyl rearrangement to form a normal peptide bond, which is thermodynamically more stable (Raghavan & Minnick, 2009). Neither peptide bond cleavage involves hydrolysis, and hydrolysis, if it occurs, converts the succinimide ring back to Asn. Once the intein is released, no further proteolytic activity occurs.|
|Molecular structure||The tertiary structure of the endonuclease has been solved (Werner et al., 2002), as well as the structure of a miniprecursor, containing fragments of the exteins (Mizutani et al., 2002). The intein in the endonuclease structure is similar to that of the hedgehog protein C-terminal domain (C46.001).|