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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1am2
Original Entry
Title:
Intein
Compound:
Mxe gyra intein
Mutant:
Yes
UniProt/Swiss-Prot:
P72065-GYRA_MYCXE
EC Class:
5.99.1.3
Other CSA Entries:
Overview of all sites for 1am2
Homologues of 1am2
Entries for UniProt/Swiss-Prot: P72065
Entries for EC: 5.99.1.3
Other Databases:
PDB entry: 1am2
PDBsum entry: 1am2
UniProt/Swiss-Prot: P72065
IntEnz entry: 5.99.1.3
Literature Report:
Introduction:
Protein splicing is a post-translational process associated with several proteins from yeast, eubacteriae and the archae. Extein ligation differentiates protein splicing from other forms of self proteolysis such as the autocleavage function of glycosylasparaginase. The first intein to be discovered was the Saccharomyces cerevisiae VMA intein found to interrupt the 69,000 Mr catalytic subunit of vaculor H+-ATPase. The GyrA intein is 198 residues in length and lacks the normal endonuclease activity.
Mechanism:
After synthesis of a nascent protein an internal protein domain (termmed the intein) is excised from the precursor and the two external polypeptides (termed exteins) are ligated together through a peptide bond.

Catalysis begins with cleavage of the N-extein-intein peptide bond by nucleophilic attack of the Cys at the intein N terminus on the immediate upstream carbonyl carbon. A tetrahedral intermediate is formed which then collapses with lysis of the C-N bond aided by donation of a proton by His75 and steric strain. The transition state is stabilised in an oxyanion hole formed by Thr72 and Asn74. Transfer of the N-extein to the side chain hydroxyl of the Thr at the N terminus of the C-extein follows. This forms a branched intermediate at the Thr residue.

Cleavage of the intein-C-extein peptide bond occurs by cyclic imide formation of the C-terminal intein Asn residue. The tetrahedral intermediate formed by nucleophilic attack of the side chain N on the carbonly group collapses productively due to donation of a proton to the leaving amine group from His197. Thus the intein is lost from the protein with a C terminal succinimide group.

The final stage of peptide bond formation may occur without the aid of catalysis. Nucleophilic attack of the newly released free N terminus of C-extein on the ester-linked N- and C-extein junction causes formation of a peptide linkage and reformation of the Thr residue.
Sites:

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
SERA 1 0Sidechain
NucleophileSubstrate
Nucleophilic attack of the carbonyl group immediately prior to it in a N-S shift. Forms a leaving group for transesterification which transfers the N-extein to the C-extein.
Evidence from paper Evidence concerns Evidence type
PubMed ID 8895558 Related protein: UniProt Q51334 Mutagenesis of residue
PubMed ID 17544410 Related protein: UniProt Q58TX5 Kinetic studies
PubMed ID 9437427 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 8895558 Related protein: UniProt Q51334 Conservation of residue
PubMed ID 8895558 Related protein: UniProt Q51334 Kinetic studies
PubMed ID 17544410 Related protein: UniProt Q58TX5 Conservation of residue
PubMed ID 9437427 Current protein Mutagenesis of residue
PubMed ID 8895558 Related protein: UniProt Q51334 Residue is covalently bound to intermediate, based on non-structural data
PubMed ID 17544410 Related protein: UniProt Q58TX5 Mutagenesis of residue
PubMed ID 9437427 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
THRA 72 137Sidechain
ElectrostaticTransition state
Steric strainSubstrate
Stabilisation of the tetrahedral intermediate formed by Cys1 attack on the preceeding residue. A hydrogen bond to this residue forces it to lie in an unstable cis arrangement.
Evidence from paper Evidence concerns Evidence type
PubMed ID 17544410 Related protein: UniProt Q58TX5 Kinetic studies
PubMed ID 17544410 Related protein: UniProt Q58TX5 Conservation of residue
PubMed ID 9437427 Current protein Conservation of residue
PubMed ID 17544410 Related protein: UniProt Q58TX5 Mutagenesis of residue
PubMed ID 9437427 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASNA 74 139Sidechain
ElectrostaticTransition state
Stabilisation of the tetrahedral intermediate formed by Cys1 attack on the previous residue.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9437427 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 75 140Sidechain
Acid/baseSubstrate
Provides a proton to the leaving amide group of the N-extein during the N-S shift.
Evidence from paper Evidence concerns Evidence type
PubMed ID 17544410 Related protein: UniProt Q58TX5 Conservation of residue
PubMed ID 9437427 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 9437427 Current protein Conservation of residue
PubMed ID 17544410 Related protein: UniProt Q58TX5 Kinetic studies
PubMed ID 17544410 Related protein: UniProt Q58TX5 Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 197 262Sidechain
Acid/baseSubstrate
Provides a proton to the leaving amide group of the C-extein during succinimide formation.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9437427 Current protein Conservation of residue
PubMed ID 17544410 Related protein: UniProt Q58TX5 Kinetic studies
PubMed ID 8895558 Related protein: UniProt Q51334 Kinetic studies
PubMed ID 8895558 Related protein: UniProt Q51334 Conservation of residue
PubMed ID 9437427 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 17544410 Related protein: UniProt Q58TX5 Mutagenesis of residue
PubMed ID 17544410 Related protein: UniProt Q58TX5 Conservation of residue
PubMed ID 8895558 Related protein: UniProt Q51334 Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASNA 198 263Sidechain, Backbone carbonyl
ElectrophileResidue
NucleophileResidue
The side chain attacks its own carbonyl group leading to cyclisation and elimination of the branched extein.
Evidence from paper Evidence concerns Evidence type
PubMed ID 8895558 Related protein: UniProt Q51334 Residue is covalently bound to intermediate, based on non-structural data
PubMed ID 17544410 Current protein Kinetic studies
PubMed ID 8895558 Related protein: UniProt Q51334 Mutagenesis of residue
PubMed ID 9437427 Current protein Conservation of residue
PubMed ID 17544410 Current protein Mutagenesis of residue
PubMed ID 8895558 Related protein: UniProt Q51334 Kinetic studies
PubMed ID 17544410 Current protein Conservation of residue
PubMed ID 9437427 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 8895558 Related protein: UniProt Q51334 Conservation of residue
Notes:
In addition there must be a conserved Ser, Thr or Cys immediately after the final conserved Asn residue of the intein for transfer of the N terminal extein to the C terminal extein.
The EC number assigned to this structure is for the gyrase activity not for the intein activity. No EC number has been assigned for the intein activity.
References:
Which EBI biological databases are available and how do I access them? EBI Site Map