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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1iu4
Original Entry
Title:
Transferase
Compound:
Microbial transglutaminase
Mutant:
No
UniProt/Swiss-Prot:
Q8KRJ2-Q8KRJ2
EC Class:
2.3.2.13
Other CSA Entries:
Overview of all sites for 1iu4
Homologues of 1iu4
Entries for UniProt/Swiss-Prot: Q8KRJ2
Entries for EC: 2.3.2.13
Other Databases:
PDB entry: 1iu4
PDBsum entry: 1iu4
UniProt/Swiss-Prot: Q8KRJ2
IntEnz entry: 2.3.2.13
Literature Report:
Introduction:
The microbial tranglutaminase, MTG, from Streptoverticllium mobaraense folds in to aplate-like shape with a deep cleft at the edge of the molecule.

Transglutaminase, TGase, catalyses an acyl transfer mechanism in which gamma-caboxyamide groups of the peptide-bound glutamine residues act as acyl donors with e-amino lysine groups as acyl acceptors.

MTG is secreted from the cytoplasm and is activated by proteolytic processing. The additional pro-sequence consisting of 45 amino acid residues at the N terminus is cleaved off, resulting in the active form, the mature enzyme.

Industrial applications of MTG include improving the physical and textural properties of protein-rich foods.
Mechanism:
MTG is involved in catalysing an acyl transfer reaction between protein glutamine and alkylamine, producing protein N(5)-alkylglutamine and ammonia.

The residues Cys 64, Asp 255 and His 274 superimpose well on the 'Cys, His, Asp' catalytic triad of other transglutaminases, such as Factor XIII. However, the relative positions of MTG His and Asp seem to be reversed relative to the Cys residue and therefore the catalytic triad is not conserved in the active site of MTG, Hence, a cysteine-like protease mechanism is proposed, in which Asp 255 plays the role of the His residue in factor XIII-like TGases.

1. Cys 64 is sufficiently exposed to the solvent and is deprotonated by water.
2. The thiolate ion of Cys 64 nucleophilically attacks an acyl donor, the carbonyl C of the substrate Gln residue side chain.
3. Electrostatic interaction of Asp 255 with water facilitates donation of a proton from Asp 255 to the resulting oxyanion from nucleophilic attack, and ammonia is released.
4. The Asp 255 gamma-carboxyl group and the His 274 imidazole ring form a hydrogen bond to allow a favourable conformation.
5. The Asp 255 oxygen is negatively charged and nucleophilically attacks a proton of a Lys residue acyl acceptor.
6. Lys 269 side chain acts as an acyl acceptor as the NH attacks the transition state acyl group.
7. Cys 64 N and Trp 272 Ne1 contribute to the stabilising oxyanion hole by forming hydrogen bonds.
8. The product is released from the oxyanion intermediate.
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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 64 140Sidechain, Backbone amide
Acid/baseWater
NucleophileSubstrate
ElectrostaticTransition state
he Cys S is deprotonated by water and the resulting thiolate ion nucleophilically attacks the acyl group of the Gln substrate side chain. Cys 64 N forms part of the transition state-stabilising oxyanion hole.
Evidence from paper Evidence concerns Evidence type
PubMed ID 12221081 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 12221081 Current protein Ligand is essential for catalysis
PubMed ID 12221081 Current protein Structural similarity to homologue of known mechanism
PubMed ID 12221081 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 255 331Sidechain
Acid/baseResidue
ElectrostaticResidue
ElectrostaticWater
lectrostatic interaction with water initiates Asp 255 to donate a proton to the oxyanion intermediate. The carboxyl group of Asp 255 forms a hydrogen bond with His 275 imidazole ring to stabilise the transition state and ensure a favourable conformation. The negatively charged side chain oxygen on Asp 255 nucleophilically attacks a proton of the Cys residue acyl group.
Evidence from paper Evidence concerns Evidence type
PubMed ID 12221081 Current protein Mutagenesis of residue
PubMed ID 12221081 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 12221081 Current protein Structural similarity to homologue of known mechanism

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
LYSA 269 345Sidechain
NucleophileSubstrate
Lys 269 side chain acts as an acyl acceptor as the NH attacks the transition state acyl group.
Evidence from paper Evidence concerns Evidence type
PubMed ID 12221081 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
TRPA 272 348Sidechain
ElectrostaticTransition state
ys 64 N and Trp 272 Ne1 contribute to the stabilising oxyanion hole by forming hydrogen bonds.
Evidence from paper Evidence concerns Evidence type
PubMed ID 12221081 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 274 350Sidechain
ElectrostaticResidue
The carboxyl group of Asp 255 forms a hydrogen bond with His 274 imidazole ring to stabilise the transition state and ensure a favourable conformation
Evidence from paper Evidence concerns Evidence type
PubMed ID 12221081 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 12221081 Current protein Structural similarity to homologue of known mechanism
Notes:
Despite the His 274 playing a reversed role in the cysteine-like protease catalytic triad, it is worthy of note that there is much speculation about the role of this residue as being essential for catalysis.

The hydrogen bond between the His 274 imidazole ring and the Asp 255 carboxyl group actually has two antagonistic effects. Even though this electrostatic interaction keeps the active site in favourable conformation, the H bond appears to decrease the nucleophilicity of the Asp residue, thus exerting a negative effect on catalytic activity. This decreases the catalytic importance of His 274.

Hence, the mechanism may be regarded as utilising a Cys-Asp catalytic diad.
References:
1
Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense.
T. Kashiwagi and K. Yokoyama and K. Ishikawa and K. Ono and D. Ejima and H. Matsui and E. Suzuki
J Biol Chem 277, (46) 44252-60, (2002).
12221081
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